Peroxotitanate- and monosodium metal-titanate compounds as inhibitors of bacterial growth.

Sodium titanates are ion-exchange materials that effectively bind a variety of metal ions over a wide pH range. Sodium titanates alone have no known adverse biological effects but metal-exchanged titanates (or metal titanates) can deliver metal ions to mammalian cells to alter cell processes in vitro. In this work, we test a hypothesis that metal-titanate compounds inhibit bacterial growth; demonstration of this principle is one prerequisite to developing metal-based, titanate-delivered antibacterial agents. Focusing initially on oral diseases, we exposed five species of oral bacteria to titanates for 24 h, with or without loading of Au(III), Pd(II), Pt(II), and Pt(IV), and measuring bacterial growth in planktonic assays through increases in optical density. In each experiment, bacterial growth was compared with control cultures of titanates or bacteria alone. We observed no suppression of bacterial growth by the sodium titanates alone, but significant (p < 0.05, two-sided t-tests) suppression was observed with metal-titanate compounds, particularly Au(III)-titanates, but with other metal titanates as well. Growth inhibition ranged from 15 to 100% depending on the metal ion and bacterial species involved. Furthermore, in specific cases, the titanates inhibited bacterial growth 5- to 375-fold versus metal ions alone, suggesting that titanates enhanced metal-bacteria interactions. This work supports further development of metal titanates as a novel class of antibacterials.

Preclinical effectiveness of a novel pulp capping material.

INTRODUCTION: The purpose of this study was to evaluate the direct pulp capping response to a novel resin-based calcium phosphate cement (RCPC). METHODS: The RCPC was placed in contact with the exposed healthy pulps of dog teeth and in a follow-up study on the healthy or inflamed pulps of ferret teeth. The inflamed ferret teeth had reversible pulpitis induced with Salmonella typhimurium lipopolysaccharides. After direct pulp capping with RCPC or visible light-curing resin-modified calcium hydroxide material (VLCCH) as a control, the restorations were bonded using a composite resin. The pulp responses and dentin repair were evaluated histologically in dog teeth after 7, 28, or 90 days and in ferret teeth after 45 days. RESULTS: Most of the RCPC-treated healthy pulps and 75% of the RCPC-treated inflamed ferret teeth had dentin healing and repair, whereas those teeth treated with VLCCH had minimal healing and dentin repair. CONCLUSIONS: The direct pulp capping of ferret and dog teeth with RCPC was associated with superior healing in comparison to VLCCH.

Phosphate: known and potential roles during development and regeneration of teeth and supporting structures.

Inorganic phosphate (P(i)) is abundant in cells and tissues as an important component of nucleic acids and phospholipids, a source of high-energy bonds in nucleoside triphosphates, a substrate for kinases and phosphatases, and a regulator of intracellular signaling. The majority of the body's P(i) exists in the mineralized matrix of bones and teeth. Systemic P(i) metabolism is regulated by a cast of hormones, phosphatonins, and other factors via the bone-kidney-intestine axis. Mineralization in bones and teeth is in turn affected by homeostasis of P(i) and inorganic pyrophosphate (PPi), with further regulation of the P(i)/PP(i) ratio by cellular enzymes and transporters. Much has been learned by analyzing the molecular basis for changes in mineralized tissue development in mutant and knock-out mice with altered P(i) metabolism. This review focuses on factors regulating systemic and local P(i) homeostasis and their known and putative effects on the hard tissues of the oral cavity. By understanding the role of P(i) metabolism in the development and maintenance of the oral mineralized tissues, it will be possible to develop improved regenerative approaches.

Bone regeneration in cranial defects previously treated with radiation.

OBJECTIVES/HYPOTHESIS: Bone reconstruction in the head and neck region is frequently performed in the context of previous radiation treatment. Thus, the effectiveness of tissue engineering approaches for regenerating bone in radiated defects needs to be determined before considering application to patients. Incomplete healing is described when using osteoinductive protein therapy alone for bone defects previously treated with radiation. We hypothesized that a different approach using ex vivo gene therapy can heal these severely compromised defects. STUDY DESIGN: Animal study using Fisher rats. METHODS: Two weeks before surgery, rats received either no radiation or a 12 Gray radiation dose to the calvarium. Syngeneic dermal fibroblasts were transduced ex vivo using an adenoviral vector containing the cDNA for bone morphogenetic protein (BMP)-7. Critical-sized calvarial defects were created, and either a transduced cell-seeded scaffold or an autologous bone graft was placed into the defect. Nonradiated defects were harvested 4 weeks later for both groups. Radiated defects treated with bone grafts were harvested at 4 weeks, and those treated with gene therapy were harvested either at 4 or 8 weeks. Gross inspection and histology were used to evaluate wound healing. RESULTS: None of the bone grafts had gross or histologic evidence of healing at the wound margins. The nonradiated gene therapy treated defects revealed gross and histologic near-100% bone regeneration by 4 weeks after surgery. By gross inspection, the radiated defects had soft tissue admixed with islands of bone at both 4 and 8 weeks. The histologic appearance revealed areas of dense bone in a nonconfluent pattern admixed with adjacent cells having the morphologic appearance of hypertrophic chondrocytes, suggesting continued endochondral ossification. CONCLUSIONS: Preoperative radiation significantly impairs the ability of BMP-7 ex vivo gene therapy to heal rat critical-sized cranial defects. This finding has significant implications for translating this tissue engineering approach to patients with cancer-related segmental bone defects.

Identification of potential modifiers of Runx2/Cbfa1 activity in C2C12 cells in response to bone morphogenetic protein-7.

Treatment with BMP-7 causes a shift in the differentiation pathway from myoblastic to osteoblastic in C2C12 mouse myoblast precursor cells in vitro. The underlying molecular mechanism is largely unknown. BMP-7 at 200 ng/ml completely inhibited myotube formation in C2C12 cells and dramatically induced alkaline phosphatase activity up to 20-fold when compared to untreated cells by day 12 in culture. The level of Runx2/Cbfa1 mRNA, a bone-specific transcription factor, was also stimulated up to 6-fold by BMP-7 with a peak at 24 h. In addition BMP-7 treatment stimulated a 55-fold increase in osteocalcin mRNA as early as 24 h after treatment. A novel finding was that the expression of the chondrocyte markers Sox9 and type II collagen was increased as well. Runx2/Cbfa1 is a molecular switch for osteoblast differentiation. To initiate the study of modulators of Runx2/Cbfa1, such as kinases and cofactors, during osteoblastic differentiation of C2C12 cells treated by BMP-7 in vitro, microarray analyses of gene expressions were performed. Microarray data suggested that a total of 882 transcripts were either up- or downregulated at least 2-fold. Cluster analyses revealed 76 genes (including ESTs) with expression patterns that paralleled Runx2/Cbfa1. Thirteen of these 76 genes were initially selected as potential transcription modulators for further study; including CCAAT/enhancer binding protein delta, distal- less homeobox 1, forkhead box F2, insulin-like growth factor binding protein 4, an ortholog of human osteoclast stimulating factor 1 and p300/CBP-associated factor. Some transcription modulators have been associated with osteoblastic differentiation or interacted with Runx2/Cbfa1. Most of them have not been extensively studied in osteoblastic differentiation and in relationship to Runx2/Cbfa1. Thus, these studies identify potential regulators for Runx2/Cbfa1 and osteoblast differentiation. In addition, our data revealed for the first time that BMP-7 not only induced the expression of osteoblastic differentiation markers but also stimulated the expression of chondroblastic markers in C2C12 cells.

Gene therapy approaches for bone regeneration.

Gene therapy represents a promising approach for delivering regenerative molecules to specific tissues including bone. Several laboratories have shown that virus-based BMP expression vectors can stimulate osteoblast differentiation and bone formation in vivo. Both in vivo and ex vivo transduction of cells can induce bone formation at ectopic and orthotopic sites. Adenovirus and direct DNA delivery of genes encoding regenerative molecules can heal critical-sized defects of cranial and long bones. Although osteogenic activity can be demonstrated for individual BMP vectors, substantial synergies may be achieved using combinatorial gene therapy to express complimentary osteogenic signals including specific combinations of BMPs or BMPs and transcription factors. Further control of the bone regeneration process may also be achieved through the use of inducible promoters that can be used to control the timing and magnitude of expression for a particular gene. Using these types of approaches, it should be possible to mimic natural processes of bone development and fracture repair and, in so doing, be able to precisely control both the amount and type of bone regenerated.

Early events: the in vitro conversion of BMP transduced fibroblasts to chondroblasts.

Strategies for localized skeletal regeneration using either cell or ex vivo gene transfer typically utilize preosteoblasts from bone or bone marrow biopsies. Our studies have demonstrated than ex vivo bone morphogenetic proteins (BMP) transduced fibroblasts convert to osteoblasts and form bone in vivo. In addition when suspended in a variety of thermoset hydrogels, these cells are capable of ectopic or orthotopic bone formation. To study the mechanism of the phenotypic conversion of BMP transduced fibroblasts, we have initiated characterization of three-dimensional (3D) cultures derived from these cell/collagen hydrogel composites. These data reveal that the BMP, but not control transduced fibroblasts, secrete BMP-7 and acquire some chondroblastic traits in 3D cultures. These traits include altered cell shape and changes in the extracellular matrix such as accumulation of cartilage proteoglycan, type II collagen, and mineral deposition by 3 weeks. These studies suggest that this culture system may be useful for elucidating early mechanistic events in the BMP-induced conversion of fibroblasts to osteoblasts.

Ex vivo gene therapy for skeletal regeneration in cranial defects compromised by postoperative radiotherapy.

Because radiation remains a common postoperative treatment for head and neck cancers, it is critical to determine whether new bone-regenerative approaches are effective for healing craniofacial defects challenged by therapeutic doses of radiation. The objective of this study was to determine whether the deleterious effects of radiotherapy could be overcome by ex vivo gene therapy to heal craniofacial defects. Rat calvarial critical-sized defects were treated with either an inlay calvarial bone graft or syngeneic dermal fibroblasts transduced ex vivo with an adenovirus engineered to express bone morphogenetic protein 7 (BMP-7), a morphogen known to stimulate bone formation. Two weeks postoperatively, either no radiation or a single 12-Gy radiation dose was delivered to the operated area and the tissue was harvested 4 weeks later. None of the inlay bone grafts healed at the wound margins of either the radiated or nonradiated sites. In contrast, bone was successfully regenerated when using an ex vivo gene therapy approach. More bone formed in the nonradiated group as determined by the percentage of defect surface covered (87 +/- 4.1 versus 65 +/- 4.7%; p = 0.003) and percentage of defect area filled by new bone (60 +/- 5.9 versus 32 +/- 2.7%; p = 0.002). Although the effects of radiation on the wound were not completely overcome by the gene therapy approach, bone regeneration was still successful despite the radiation sensitivity of the fibroblasts. These results indicate that BMP-7 ex vivo gene therapy is capable of successfully regenerating bone in rat calvarial defects even after a therapeutic dose of radiation. This approach may represent a new strategy for regenerating skeletal elements lost due to head and neck cancer.

Bone morphogenetic protein 7 ex vivo gene therapy.

Acquired and congenital skeletal defects arise from trauma and developmental abnormalities as well as ablative cancer surgery. Reconstructive surgery to repair the functional limitations and aesthetic concerns caused by these defects can be complex and extensive, and therapeutic restoration of structure and function is often difficult. Currently, autologous bone grafts are the "gold standard" method for surgical repair. Protein therapy, whereby recombinant or naturally sourced bone morphogenetic proteins are surgically implanted in a defect, has recently been approved for limited clinical use. Yet protein therapy and other methods including allografting, alloplastic implants and cell therapy have significant limitations. Recent advances in bone morphogenetic protein 7 ex vivo gene therapy for localized skeletal regeneration address these limitations.

Bone morphogenetic protein-transduced human fibroblasts convert to osteoblasts and form bone in vivo.

Experimental cell or ex vivo gene therapy for localized bone formation typically uses osteoprogenitor cells propagated from periosteum or bone marrow. Both require bone or marrow biopsies to obtain cells. We have demonstrated that implantation of gingival or dermal fibroblasts transduced with BMP ex vivo, using a recombinant adenovirus (AdCMVBMP) attached to porous biodegradable scaffolds, form bone in vivo. Here we show that BMP-7-transduced fibroblasts suspended in injectable thermoset hydrogels form complete ossicles on subcutaneous injection and repair segmental defects in rat femurs. Bone formation was preceded by an intermediate cartilage stage. To determine the fate of the implanted transduced cells, thermoset hydrogel suspensions of ex vivo BMP-7-transduced or nontransduced fibroblasts were placed in diffusion chambers and implanted to allow development in vivo without direct contact with host cells. Only the BMP-transduced fibroblasts formed bone within the diffusion chambers in vivo, revealing that BMP transduction induces osteoblastic conversion of these cells.