Podoplanin is dispensable for mineralized tissue formation and maintenance in the Swiss outbred mouse background.

Podoplanin, PDPN, is a mucin-type transmembrane glycoprotein widely expressed in many tissues, including lung, kidney, lymph nodes, and mineralized tissues. Its function is critical for lymphatic formation, differentiation of type I alveolar epithelial lung cells, and for bone response to biomechanical loading. It has previously been shown that Pdpn null mice die at birth due to respiratory failure emphasizing the importance of Pdpn in alveolar lung development. During the course of generation of Pdpn mutant mice, we found that most Pdpn null mice in the 129S6 and C57BL6/J mixed genetic background die at the perinatal stage, similar to previously published studies with Pdpn null mice, while all Pdpn null mice bred with Swiss outbred mice survived. Surviving mutant mice in the 129S6 and C57BL6/J mixed genetic background showed alterations in the osteocyte lacunocanalicular network, especially reduced osteocyte canaliculi in the tibial cortex with increased tibial trabecular bone. However, adult Pdpn null mice in the Swiss outbred background showed no overt differences in their osteocyte lacunocnalicular network, bone density, and no overt differences when challenged with exercise. Together, these data suggest that genetic variations present in the Swiss outbred mice compensate for the loss of function of PDPN in lung, kidney, and bone.

Ambulatory management of primary spontaneous pneumothorax: an open-label, randomised controlled trial.

BACKGROUND: Primary spontaneous pneumothorax occurs in otherwise healthy young patients. Optimal management is not defined and often results in prolonged hospitalisation. Data on efficacy of ambulatory options are poor. We aimed to describe the duration of hospitalisation and safety of ambulatory management compared with standard care. METHODS: In this open-label, randomised controlled trial, adults (aged 16-55 years) with symptomatic primary spontaneous pneumothorax were recruited from 24 UK hospitals during a period of 3 years. Patients were randomly assigned (1:1) to treatment with either an ambulatory device or standard guideline-based management (aspiration, standard chest tube insertion, or both). The primary outcome was total length of hospital stay including re-admission up to 30 days after randomisation. Patients with available data were included in the primary analysis and all assigned patients were included in the safety analysis. The trial was prospectively registered with the International Standard Randomised Clinical Trials Number, ISRCTN79151659. FINDINGS: Of 776 patients screened between July, 2015, and March, 2019, 236 (30%) were randomly assigned to ambulatory care (n=117) and standard care (n=119). At day 30, the median hospitalisation was significantly shorter in the 114 patients with available data who received ambulatory treatment (0 days [IQR 0-3]) than in the 113 with available data who received standard care (4 days [IQR 0-8]; p<0·0001; median difference 2 days [95% CI 1-3]). 110 (47%) of 236 patients had adverse events, including 64 (55%) of 117 patients in the ambulatory care arm and 46 (39%) of 119 in the standard care arm. All 14 serious adverse events occurred in patients who received ambulatory care, eight (57%) of which were related to the intervention, including an enlarging pneumothorax, asymptomatic pulmonary oedema, and the device malfunctioning, leaking, or dislodging. INTERPRETATION: Ambulatory management of primary spontaneous pneumothorax significantly reduced the duration of hospitalisation including re-admissions in the first 30 days, but at the expense of increased adverse events. This data suggests that primary spontaneous pneumothorax can be managed for outpatients, using ambulatory devices in those who require intervention. FUNDING: UK National Institute for Health Research.

Characterization of new bone morphogenetic protein (Bmp)-2 regulatory alleles.

Bone morphogenetic protein 2 (BMP2, HGNC:1069, GeneID: 650) is a classical morphogen; a molecule that acts at a distance and whose concentration influences cell proliferation, differentiation, and apoptosis. Key events requiring precise Bmp2 regulation include heart specification and morphogenesis and neural development. In mesenchymal cells, the concentration of BMP2 influences myogenesis, adipogenesis, chondrogenesis, and osteogenesis. Because the amount, timing, and location of BMP2 synthesis influence pattern formation and organogenesis, the mechanisms that regulate Bmp2 are crucial. A sequence within the 3'UTR of the Bmp2 mRNA termed the "ultra-conserved sequence" (UCS) has been largely unchanged since fishes and mammals diverged. Cre-lox mediated deletion of the UCS in a reporter transgene revealed that the UCS may repress Bmp2 in proepicardium, epicardium, and epicardium-derived cells (EPDC) and in tissues with known epicardial contributions (coronary vessels and valves). The UCS also repressed the transgene in the aorta, outlet septum, posterior cardiac plexus, cardiac and extra-cardiac nerves, and neural ganglia. We used homologous recombination and conditional deletion to generate three new alleles in which the Bmp2 3'UTR was altered as follows: a UCS flanked by loxP sites with or without a neomycin resistance targeting vector, or a deleted UCS. Deletion of the UCS was associated with elevated Bmp2 mRNA and BMP signaling levels, reduced fitness, and embryonic malformations.

Reducing the Incidence of Cast-related Skin Complications in Children Treated With Cast Immobilization.

BACKGROUND: Cast immobilization remains the mainstay of pediatric orthopaedic care, yet little is known about the incidence of cast-related skin complications in children treated with cast immobilization. The purposes of this quality improvement project were to: (1) establish a baseline rate of cast-related skin complications in children treated with cast immobilization, (2) identify trends in children who experienced cast-related skin complications, (3) design an intervention aimed at decreasing the rate of cast-related skin complications, and (4) determine the effectiveness of the intervention. METHODS: A prospective interrupted time-series design was used to determine the incidence of cast-related skin complications overtime and compare the rates of skin complications before and after an intervention designed to decrease the incidence of cast-related heel complications. All consecutive patients who were treated with cast immobilization from September 2012 to September 2014 were included. A cast-related skin complications data collection tool was used to capture all cast-related skin complications. A high rate of heel events was noted in our preliminary analysis and an intervention was designed to decrease the rate of cast-related skin complications, including the addition of padding during casting and respective provider education. RESULTS: The estimated cast-related skin events rate for all patients was 8.9 per 1000 casts applied. The rate for the total preintervention sample was 13.6 per 1000 casts which decreased to 6.6 in the postintervention sample. When examining the heel-only group, the rate was 17.1 per 1000 lower extremity casts applied in the preintervention group and 6.8 in the postintervention group. CONCLUSIONS: Incorporating padding to the heel of lower extremity cast was an effective intervention in decreasing the incidence of cast-related skin complications in patients treated with cast immobilization. LEVEL OF EVIDENCE: Level II.

Bmp2 in osteoblasts of periosteum and trabecular bone links bone formation to vascularization and mesenchymal stem cells.

We generated a new Bmp2 conditional-knockout allele without a neo cassette that removes the Bmp2 gene from osteoblasts (Bmp2-cKO(ob)) using the 3.6Col1a1-Cre transgenic model. Bones of Bmp2-cKO(ob) mice are thinner, with increased brittleness. Osteoblast activity is reduced as reflected in a reduced bone formation rate and failure to differentiate to a mature mineralizing stage. Bmp2 in osteoblasts also indirectly controls angiogenesis in the periosteum and bone marrow. VegfA production is reduced in Bmp2-cKO(ob) osteoblasts. Deletion of Bmp2 in osteoblasts also leads to defective mesenchymal stem cells (MSCs), which correlates with the reduced microvascular bed in the periosteum and trabecular bones. Expression of several MSC marker genes (α-SMA, CD146 and Angiopoietin-1) in vivo, in vitro CFU assays and deletion of Bmp2 in vitro in α-SMA(+) MSCs support our conclusions. Critical roles of Bmp2 in osteoblasts and MSCs are a vital link between bone formation, vascularization and mesenchymal stem cells.

PedsQL correlates to PODCI in pediatric orthopaedic outpatient clinic.

PURPOSE: Quality-of-life (QOL) measures can be a valuable tool to assess the general welfare across a spectrum of patients in a pediatric orthopaedic outpatient clinic and can be a simple way to assess patient-based outcomes particularly for quality initiatives. The Pediatric Outcomes Data Collection Instrument (PODCI) is validated for many orthopaedic conditions but typically takes around 20 minutes to complete (86 questions). The Pediatric Quality of Life Inventory (PedsQL) takes <4 minutes to complete (23 questions) but has not been assessed in an orthopaedic setting. We initiated this study to find the best method for assessing QOL in our outpatient clinic. A short pediatric QOL measure that is correlated to an established orthopaedic-specific QOL measure is needed; therefore, we compared the PedsQL to the PODCI in the outpatient orthopaedic clinic. METHODS: This was a quality initiative project and as such did not require a priori IRB approval. Families of patients 2 to 18 years old who presented for follow-up after upper or lower extremity fractures or brachial plexus injuries in the orthopaedic clinic from October 2010 through August 2011 were asked to fill out both the PODCI and the PedsQL. Patients aged 5 years and older filled out a patient-report PedsQL; patients aged 11 years and older filled out the patient-report PODCI. Parents/guardians completed questionnaires for children of all ages. Most fracture patients (and/or their parent/guardian) repeated the questionnaires after 6 to 12 weeks. Data were then assessed for correlation between the PODCI and PedsQL. RESULTS: A total of 428 parent/guardian reports for 283 patients and 172 self-reports for 104 patients were included. The correlation between the PODCI Global score and the PedsQL Total score for the parent/guardian-reported questionnaires for all injuries was 0.77 (95% confidence interval, 0.72-0.82). When categorized within domains and injuries, parent/guardian-reported correlations ranged from 0.23 to 0.79. In patients aged 11 years and older, the correlation between the PODCI and PedsQL for the patient-reported questionnaire for all injuries was 0.85 (95% confidence interval, 0.80-0.89). When categorized within domains and injuries, patient-reported correlations ranged from 0.30 to 0.99. CONCLUSIONS: Utilizing the substantially shorter PedsQL in a high volume orthopaedic clinic as a substitute for the PODCI for quality improvement measures seems reasonable. Correlation between the PedsQL Global score and the PODCI Total score for orthopaedic patients is strong. Utilizing the patient-reported questionnaires when age appropriate is best. In this era of increased outcome reporting, PedsQL may be a valuable tool.

Role of bone morphogenetic proteins on cochlear hair cell formation: analyses of Noggin and Bmp2 mutant mice.

The mammalian organ of Corti of the inner ear is a highly sophisticated sensory end organ responsible for detecting sound. Noggin is a secreted glycoprotein, which antagonizes bone morphogenetic proteins 2 and 4 (Bmp2 and Bmp4). The lack of this antagonist causes increased rows of inner and outer hair cells in the organ of Corti. In mice, Bmp2 is expressed transiently in nascent cochlear hair cells. To investigate whether Noggin normally modulates the levels of Bmp2 for hair cell formation, we deleted Bmp2 in the cochlear hair cells using two cre strains, Foxg1(cre/+) and Gfi1(cre/+). Bmp2 conditional knockout cochleae generated using these two cre strains show normal hair cells. Furthermore, Gfi1(cre/+);Bmp2(lox/-) mice are viable and have largely normal hearing. The combined results of Noggin and Bmp2 mutants suggest that Noggin is likely to regulate other Bmps in the cochlea such as Bmp4.

Climate Disruption and Personal Health-A Conversation About Watershed: Attending to Body and Earth in Distress.

In her book Watershed: Attending to Body and Earth in Distress (2021, University of Minnesota Press), author Ranae Lenor Hanson poses the question: What if we cared for the strained earth as tenderly as we care for a human body in a medical crisis? Using the metaphor of diabetes to explore the climate crisis, Hanson weaves stories from her climate-refugee and urban climate-affected students with those of Minnesota watersheds that have provided a nurturing web for her life. In this article, four former students whose stories appear in the book reflected on their reactions to Watershed.

E11/gp38 selective expression in osteocytes: regulation by mechanical strain and role in dendrite elongation.

Within mineralized bone, osteocytes form dendritic processes that travel through canaliculi to make contact with other osteocytes and cells on the bone surface. This three-dimensional syncytium is thought to be necessary to maintain viability, cell-to-cell communication, and mechanosensation. E11/gp38 is the earliest osteocyte-selective protein to be expressed as the osteoblast differentiates into an osteoid cell or osteocyte, first appearing on the forming dendritic processes of these cells. Bone extracts contain large amounts of E11, but immunostaining only shows its presence in early osteocytes compared to more deeply embedded cells, suggesting epitope masking by mineral. Freshly isolated primary osteoblasts are negative for E11 expression but begin to express this protein in culture, and expression increases with time, suggesting differentiation into the osteocyte phenotype. Osteoblast-like cell lines 2T3 and Oct-1 also show increased expression of E11 with differentiation and mineralization. E11 is highly expressed in MLO-Y4 osteocyte-like cells compared to osteoblast cell lines and primary osteoblasts. Differentiated, mineralized 2T3 cells and MLO-Y4 cells subjected to fluid flow shear stress show an increase in mRNA for E11. MLO-Y4 cells show an increase in dendricity and elongation of dendrites in response to shear stress that is blocked by small interfering RNA specific to E11. In vivo, E11 expression is also increased by a mechanical load, not only in osteocytes near the bone surface but also in osteocytes more deeply embedded in bone. Maximal expression is observed not in regions of maximal strain but in a region of potential bone remodeling, suggesting that dendrite elongation may be occurring during this process. These data suggest that osteocytes may be able to extend their cellular processes after embedment in mineralized matrix and have implications for osteocytic modification of their microenvironment.

Characterization of a novel murine Sost ERT2 Cre model targeting osteocytes.

Transgenic mice are widely used to delete or overexpress genes in a cell specific manner to advance knowledge of bone biology, function and disease. While numerous Cre models exist to target gene recombination in osteoblasts and osteoclasts, few target osteocytes specifically, particularly mature osteocytes. Our goal was to create a spatial and temporal conditional Cre model using tamoxifen to induce Cre activity in mature osteocytes using a Bac construct containing the 5' and 3' regions of the Sost gene (Sost ERT2 Cre). Four founder lines were crossed with the Ai9 Cre reporter mice. One founder line showed high and specific activity in mature osteocytes. Bones and organs were imaged and fluorescent signal quantitated. While no activity was observed in 2 day old pups, by 2 months of age some osteocytes were positive as osteocyte Cre activity became spontaneous or 'leaky' with age. The percentage of positive osteocytes increased following tamoxifen injection, especially in males, with 43% to 95% positive cells compared to 19% to 32% in females. No signal was observed in any bone surface cell, bone marrow, nor in muscle with or without tamoxifen injection. No spontaneous signal was observed in any other organ. However, with tamoxifen injection, a few positive cells were observed in kidney, eye, lung, heart and brain. All other organs, 28 in total, were negative with tamoxifen injection. However, with age, a muscle phenotype was apparent in the Sost-ERT2 Cre mice. Therefore, although this mouse model may be useful for targeting gene deletion or expression to mature osteocytes, the muscle phenotype may restrict the use of this model to specific applications and should be considered when interpreting data.

Generation of a conditional null allele for Dmp1 in mouse.

Dentin matrix protein1 (DMP1), highly conserved in humans and mice, is highly expressed in teeth, the skeleton, and to a lesser extent in nonskeletal tissues such as brain, kidney, and salivary gland. Pathologically, DMP1 is associated with several forms of cancers and with tumor-induced osteomalacia. Conventional disruption of the murine Dmp1 gene results in defects in dentin in teeth and in the skeleton, including hypophosphatemic rickets, and abnormalities in phosphate homeostasis. Human DMP1 mutations are responsible for the condition known as autosomal recessive hypophosphatemic rickets. For better understanding of the roles of DMP1 in different tissues at different stages of development and in pathological conditions, we generated Dmp1 floxed mice in which loxP sites flank exon 6 that encodes for over 80% of DMP1 protein. We demonstrate that Cre-mediated recombination using Sox2-Cre, a Cre line expressed in epiblast during early embryogenesis, results in early deletion of the gene and protein. These homozygous Cre-recombined null mice display an identical phenotype to conventional null mice. This animal model will be useful to reveal distinct roles of DMP1 in different tissues at different ages.

Cx43 overexpression in osteocytes prevents osteocyte apoptosis and preserves cortical bone quality in aging mice.

Young, skeletally mature mice lacking Cx43 in osteocytes exhibit increased osteocyte apoptosis and decreased bone strength, resembling the phenotype of old mice. Further, the expression of Cx43 in bone decreases with age, suggesting a contribution of reduced Cx43 levels to the age-related changes in the skeleton. We report herein that Cx43 overexpression in osteocytes achieved by using the DMP1-8kb promoter (Cx43OT mice) attenuates the skeletal cortical, but not trabecular bone phenotype of aged, 14-month-old mice. The percentage of Cx43-expressing osteocytes was higher in Cx43OT mice, whereas the percentage of Cx43 positive osteoblasts remained similar to wild type (WT) littermate control mice. The percentage of apoptotic osteocytes and osteoblasts was increased in aged WT mice compared to skeletally mature, 6-month-old WT mice, and the percentage of apoptotic osteocytes, but not osteoblasts, was decreased in age-matched Cx43OT mice. Aged WT mice exhibited decreased bone formation and increased bone resorption as quantified by histomorphometric analysis and circulating markers, compared to skeletally mature mice. Further, aged WT mice exhibited the expected decrease in bone biomechanical structural and material properties compared to young mice. Cx43 overexpression prevented the increase in osteoclasts and decrease in bone formation on the endocortical surfaces, and the changes in circulating markers in the aged mice. Moreover, the ability of bone to resist damage was preserved in aged Cx43OT mice both at the structural and material level. All together, these findings suggest that increased Cx43 expression in osteocytes ameliorates age-induced cortical bone changes by preserving osteocyte viability and maintaining bone formation, leading to improved bone strength.

Noise Reduction to Reduce Patient Anxiety During Cast Removal: Can We Decrease Patient Anxiety With Cast Removal by Wearing Noise Reduction Headphones During Cast Saw Use?

BACKGROUND: Noise reduction headphones decrease the sound during cast removal. Their effectiveness in decreasing anxiety has not been studied. PURPOSE: Compare pediatric patients' anxiety levels during cast removal with and without utilization of noise reduction headphones combined with use of a personal electronic device. METHODS: Quality improvement project. Patients randomly assigned to noise reduction headphone group or standard care group during cast removal. Faces, Legs, Activity, Cry, and Consolability Scale and heart rate were evaluated prior to, during, and after cast removal. Data were compared across groups. RESULTS: Fifty patients were included; 25 per group. No difference detected between the 2 groups in Faces, Legs, Activity, Cry, and Consolability Scale score prior to (p = .05) or after cast removal (p = .30). During cast removal, the headphone group had lower FLACC Scale scores (p = .03). Baseline heart rate was lower in the headphone group prior to (p = .02) and after (p = .005) cast removal with no difference during cast removal (p = .24). CONCLUSION: Utilizing noise reduction headphones and a personal electronic device during the cast removal process decreases patient anxiety.

Old age and the associated impairment of bones' adaptation to loading are associated with transcriptomic changes in cellular metabolism, cell-matrix interactions and the cell cycle.

In old animals, bone's ability to adapt its mass and architecture to functional load-bearing requirements is diminished, resulting in bone loss characteristic of osteoporosis. Here we investigate transcriptomic changes associated with this impaired adaptive response. Young adult (19-week-old) and aged (19-month-old) female mice were subjected to unilateral axial tibial loading and their cortical shells harvested for microarray analysis between 1h and 24h following loading (36 mice per age group, 6 mice per loading group at 6 time points). In non-loaded aged bones, down-regulated genes are enriched for MAPK, Wnt and cell cycle components, including E2F1. E2F1 is the transcription factor most closely associated with genes down-regulated by ageing and is down-regulated at the protein level in osteocytes. Genes up-regulated in aged bone are enriched for carbohydrate metabolism, TNFα and TGFß superfamily components. Loading stimulates rapid and sustained transcriptional responses in both age groups. However, genes related to proliferation are predominantly up-regulated in the young and down-regulated in the aged following loading, whereas those implicated in bioenergetics are down-regulated in the young and up-regulated in the aged. Networks of inter-related transcription factors regulated by E2F1 are loading-responsive in both age groups. Loading regulates genes involved in similar signalling cascades in both age groups, but these responses are more sustained in the young than aged. From this we conclude that cells in aged bone retain the capability to sense and transduce loading-related stimuli, but their ability to translate acute responses into functionally relevant outcomes is diminished.

Transcriptional regulation of BMP-2 activated genes in osteoblasts using gene expression microarray analysis: role of Dlx2 and Dlx5 transcription factors.

This presentation will focus on using microarray data on a clonal osteoblast cell model to analyze the early BMP-2 responsive genes, as well as some of the later genes regulated by BMP2 during different phases of mineralization. We will focus on the early phases of gene expression that occur after BMP2 signaling from 30 min up to 1 day. The hypothesis is that understanding how these early genes are regulated during the initial multilayering and growth phase of osteoblasts will lead to models of how BMP activity stimulates cell growth, cell migration, multilayering, matrix deposition and remodeling phase that allows subsequent mineralization. The Dlx2 and Dlx5 homeobox genes have been shown to be critical for bone formation both in vitro and in vivo. Both Dlx 2 and Dlx5 are activated within 15-30 minutes after BMP2 addition to the mouse 2T3 osteoblast model and primary fetal rat calvarial osteoblasts. The Dlx2 and Dlx5 genes stay elevated in the presence of BMP2 for up to 5 days, a time when overt mineralization is just beginning. To understand the genomic network that Dlx5 and Dlx2 regulate at the transcription level, we have taken an approach where we use a specific transcription repressor protein, Engrailed, ligated to the Dlx5 homeodomain. The idea is that this Eng-Dlx5 protein will interact with Dlx5 and possibly Dlx2 and related Dlx- regulated genes in vivo and down-regulate their transcriptional initiation. Using a microarray approach with over 5,000 known genes we can identify the genes that are directly and indirectly regulated by Dlx5 and Dlx2. This will allow us to build an initial genomic network of Dlx- regulated genes at the transcriptional level. We will present our model and preliminary efforts at understanding the genomic network regulated by this important BMP2-regulated transcription factor class in osteoblast biology.

Opportunities lost and found: experiences of patients with cerebral palsy and their parents transitioning from pediatric to adult healthcare.

PURPOSE: To describe and define the experiences of adults with cerebral palsy (CP) and parents of adults with CP who have been involved in a transfer of physiatry care from pediatric to adult healthcare and to explore their experiences more generally in the transition from pediatric to adult services. METHODS: A qualitative research approach was used. Semi-structured focus group interviews were conducted with adults with CP (n=5) and parents of adults with CP (n=8) to explore the health care transition (HCT) process from pediatric to adult healthcare. Four key content domains were used to facilitate the focus groups; 1) Transition Planning, 2) Accessibility of Services, 3) Experience with Adult Providers, and 4) Recommendations for Improving the Transition Process. Conventional content analysis was used to analyze the data. RESULTS: Four themes emerged from the focus groups; Lost in Transition, Roadmap to Care, List of None, and One Stop Shopping. Participants felt lost in the HCT process, requested a transparent transition plan, expressed concern regarding access to adult healthcare, and made recommendations for improvements. CONCLUSION: Challenges in transitioning from pediatric to adult health care were identified by all participants and several strategies were recommended for improvement.

Epidemiology and prevention of cast saw injuries: results of a quality improvement program at a single institution.

BACKGROUND: An investigation was conducted to establish the hospital-wide prevalence of cast saw injuries and to identify variables that put patients at increased risk, with the goal of reducing the injury rate. METHODS: Information was collected from January 2010 through December 2012 on all patients who had a cast removed or cut at our institution. Locations included the operating suites, emergency department, ambulatory clinics, and hospital floors. A cast cutting log was used to capture the total number of casts cut. An adverse event form was used to document each injury. A continuous quality improvement approach was used throughout the study period to implement incremental improvements to our program. Changes included an education and certification program on cast saw use for all providers, a protocol for a plastic surgery consultation, and a cast saw blade inspection protocol with maintenance logs. RESULTS: Twenty-nine injuries occurred in 23,615 cast cuttings over the three years, for an overall rate of 1.23 (95% confidence interval [CI], 0.86 to 1.76) per 1000. A minor decrease in cast saw injuries was recorded over the course of the study (eleven of 8043 [1.37 per 1000] in 2010, ten of 7885 [1.27 per 1000] in 2011, and eight of 7687 [1.04 per 1000] in 2012), but the decrease was not significant (p = 0.87). The emergency department had the highest rate of cast saw injuries (p < 0.0001), with a significantly greater rate during the night compared with the day (eleven of 1293 [8.51 per 1000] compared with fifteen of 19,419 [0.77 per 1000], respectively; p < 0.0001). The injuries were all minor. Key risk factors for a cast saw injury included provider inexperience, patient sedation, and poor cast saw blade condition. CONCLUSIONS: The rate of cast saw injuries in a busy pediatric orthopaedic department was small, but a considerably increased risk existed for those patients cared for in the emergency department by orthopaedic residents. Improving education and training in cast saw use has the potential to decrease the prevalence of cast saw injuries over time.

Enamel matrix derivative: protein components and osteoinductive properties.

BACKGROUND: Although enamel matrix derivative (EMD) has demonstrated the ability to promote angiogenesis and osteogenesis both in vitro and in vivo, the specific elements within the EMD compound responsible for these effects remain unknown. METHODS: Nine different protein pools from a commercially produced EMD were collected based on molecular weight. Six of these pools, along with the complete EMD unfractionated compound and positive and negative controls, were tested for their ability to induce bone formation in a calvarial induction assay. Immunocytochemistry of phosphorylated SMAD1/5/8 (phospho-SMAD), osterix, and vascular endothelial growth factor A (VEGF-A) was carried out at selected time points. Finally, proteomic analysis was completed to determine the specific protein-peptide content of the various osteoinductive pools. RESULTS: One of the lower-molecular-weight pools tested, pool 7, showed bone induction responses significantly greater than those of the other pools and the complete EMD compound and was concentration dependent. Dynamic bone formation rate analysis demonstrated that pool 7 was optimally active at the 5- to 10-µg concentration. It was demonstrated that EMD and pool 7 induced phospho-SMAD, osterix, and VEGF-A, which is indicative of increased bone morphogenetic protein (BMP) signaling. Proteomic composition analysis demonstrated that pool 7 had the highest concentration of the biologically active amelogenin-leucine-rich amelogenin peptide and ameloblastin 17-kDa peptides. CONCLUSIONS: These studies demonstrate that the low-molecular-weight protein pools (7 to 17 kDa) within EMD have greater osteoinductive potential than the commercially available complete EMD compound and that the mechanism of action, in part, is through increased BMP signaling and increased osterix and VEGF-A. With this information, selected components of EMD can now be formulated for optimal osteo- and angio-genesis.

In vitro and in vivo approaches to study osteocyte biology.

Osteocytes, the most abundant cell population of the bone lineage, have been a major focus in the bone research field in recent years. This population of cells that resides within mineralized matrix is now thought to be the mechanosensory cell in bone and plays major roles in the regulation of bone formation and resorption. Studies of osteocytes had been impaired by their location, resulting in numerous attempts to isolate primary osteocytes and to generate cell lines representative of the osteocytic phenotype. Progress has been achieved in recent years by utilizing in vivo genetic technology and generation of osteocyte directed transgenic and gene deficiency mouse models. We will provide an overview of the current in vitro and in vivo models utilized to study osteocyte biology. We discuss generation of osteocyte-like cell lines and isolation of primary osteocytes and summarize studies that have utilized these cellular models to understand the functional role of osteocytes. Approaches that attempt to selectively identify and isolate osteocytes using fluorescent protein reporters driven by regulatory elements of genes that are highly expressed in osteocytes will be discussed. In addition, recent in vivo studies utilizing overexpression or conditional deletion of various genes using dentin matrix protein (Dmp1) directed Cre recombinase are outlined. In conclusion, evaluation of the benefits and deficiencies of currently used cell lines/genetic models in understanding osteocyte biology underlines the current progress in this field. The future efforts will be directed towards developing novel in vitro and in vivo models that would additionally facilitate in understanding the multiple roles of osteocytes.

Bone morphogenetic protein-2 gene controls tooth root development in coordination with formation of the periodontium.

Formation of the periodontium begins following onset of tooth-root formation in a coordinated manner after birth. Dental follicle progenitor cells are thought to form the cementum, alveolar bone and Sharpey's fibers of the periodontal ligament (PDL). However, little is known about the regulatory morphogens that control differentiation and function of these progenitor cells, as well as the progenitor cells involved in crown and root formation. We investigated the role of bone morphogenetic protein-2 (Bmp2) in these processes by the conditional removal of the Bmp2 gene using the Sp7-Cre-EGFP mouse model. Sp7-Cre-EGFP first becomes active at E18 in the first molar, with robust Cre activity at postnatal day 0 (P0), followed by Cre activity in the second molar, which occurs after P0. There is robust Cre activity in the periodontium and third molars by 2 weeks of age. When the Bmp2 gene is removed from Sp7(+) (Osterix(+)) cells, major defects are noted in root, cellular cementum and periodontium formation. First, there are major cell autonomous defects in root-odontoblast terminal differentiation. Second, there are major alterations in formation of the PDLs and cellular cementum, correlated with decreased nuclear factor IC (Nfic), periostin and α-SMA(+) cells. Third, there is a failure to produce vascular endothelial growth factor A (VEGF-A) in the periodontium and the pulp leading to decreased formation of the microvascular and associated candidate stem cells in the Bmp2-cKO(Sp7-Cre-EGFP). Fourth, ameloblast function and enamel formation are indirectly altered in the Bmp2-cKO(Sp7-Cre-EGFP). These data demonstrate that the Bmp2 gene has complex roles in postnatal tooth development and periodontium formation.