Morphologic analysis of BMP-9 gene therapy-induced osteogenesis.

The present study was performed to determine the histological, ultrastructural, and radiographic changes that occur over time at intramuscular BMP-9 gene therapy treatment sites. Several members of the bone morphogenetic protein (BMP) family have the potential to induce osteochondrogenesis when the protein is delivered to rodents, canines, rabbits, and nonhuman primates. Previous studies have also demonstrated that BMP gene therapy utilizing adenoviral vectors can also stimulate orthotopic and heterotopic bone formation in rodents and rabbits. Athymic nude and Sprague-Dawley rats were injected with Ad-BMP-9 or Ad-beta-Gal (3.75 x 10(9) particles) in their thigh musculature and light microscopic, electron microscopic, and computerized tomography analysis was performed 3, 6, 9, 12, 15, 18, 21, and 100 days later. To assess early mesenchymal cell proliferation, a bromodeoxyuridine (BrdU) immunohistochemical analysis was also performed 48, 60, and 72 hr postinjection in athymic nude rats. All animals demonstrated extensive endochondral bone formation at the Ad-BMP-9 treatment sites within 3 weeks. The Sprague-Dawley rats also exhibited a massive, acute inflammatory infiltrate during the first week. Proliferating mesenchymal stem cells were clearly evident as early as 2 days after treatment, which differentiated into small or hypertrophied chondrocytes during the next week. During the third week, the cartilaginous matrix mineralized and formed woven bone, which converted to lamellar bone by 3 months. No evidence of bone formation was demonstrated at the Ad-beta-Gal injection sites in the athymic nude or Sprague-Dawley rats. In addition, no cellular proliferation was seen at the Ad-beta-Gal treatment sites in the athymic nude animals as assessed by light microscopy and BrdU immunohistochemistry. The extensive bone formation induced by Ad-BMP-9 suggests that BMP gene therapy may have potential utility in the treatment of degenerative, rheumatic, or traumatic bone pathology.

A light and electron microscopic study of ectopic tendon and ligament formation induced by bone morphogenetic protein-13 adenoviral gene therapy.

OBJECT: Bone morphogenetic proteins (BMPs) are involved in the growth and development of many tissues, but it is their role in skeletal development and their unique ability to induce ectopic and orthotopic osteogenesis that have attracted the greatest interest. Expression of the BMP-13 gene is predominantly localized to hypertrophic chondrocytes in regions of endochondral bone formation during development, as well as in mature articular cartilage in the adult. In addition, the application of BMP-13 on a collagen carrier induces neotendon/neoligament formation when delivered subcutaneously or intramuscularly in rodents. The aim of the present study was to determine the histological and ultrastructural changes that occur after the intramuscular injection of a first-generation BMP-13 adenoviral vector. METHODS: Athymic nude rats were injected with 3.75 x 10(10) plaque-forming units of adenovirus (Ad)-BMP-13 or Ad-beta-galactosidase in the thigh musculature, and the region was examined using light and electron microscopy at various time points between 2 days and 100 days postinjection. As early as 2 days after injection of Ad-BMP-13, progenitor cells were observed infiltrating between the transduced muscle fibers. These cells subsequently proliferated, differentiated, and secreted large amounts of collagenous extracellular matrix. By 100 days postinjection, the treated tissue displayed the histological and ultrastructural appearance of neotendon/neoligament, which was clearly demarcated from the surrounding muscle. Small foci of bone and fibrocartilage were also seen within the treated tissue. A short-term bromodeoxyuridine study also demonstrated rapid mesenchymal cell proliferation at the Ad-BMP-13 injection site as early as 48 hours postinjection. At all time points, the control AD-beta-gal injection sites were found to contain only normal muscle, without evidence of inflammation or mesenchymal cell proliferation. CONCLUSIONS: The results of this study indicate that in the future the use of the BMP-13 gene may have therapeutic utility for the healing of tendon and ligament tears and avulsion injuries.

Use of bone morphogenetic protein-9 gene therapy to induce spinal arthrodesis in the rodent.

OBJECT: Bone morphogenetic proteins (BMPs) have been shown to have significant osteoinductive activity in numerous in vitro and in vivo assay systems, and BMP-2 and BMP-7 are currently being evaluated in human clinical studies. In the spinal region, BMPs have been shown to promote spinal arthrodesis at a higher rate than autologous bone alone. The delivery of BMPs via direct or ex vivo gene therapy techniques is also currently being evaluated and has shown promise in several mammalian models. The present study was designed to evaluate the efficacy of the use of direct, percutaneous BMP-9 adenoviral gene therapy to promote spinal fusion in the rodent. METHODS: Each animal was injected with 7.5x10(8) pfu of a BMP-9 adenoviral vector in the lumbar paraspinal musculature and allowed to survive 16 weeks. Computerized tomography studies and histological analysis demonstrated massive bone induction at the injection sites, clearly leading to solid spinal arthrodesis, without evidence of pseudarthroses, nerve root compression, or systemic side effects. CONCLUSIONS: The results of this study strongly support the advancement of BMP gene therapy techniques toward clinical use.

In vivo endochondral bone formation using a bone morphogenetic protein 2 adenoviral vector.

Bone morphogenetic proteins (BMPs) are polypeptides that induce ectopic bone formation in standard rat in vivo assay systems. Previous studies have demonstrated the clinical utility of these proteins in spinal fusion, fracture healing, and prosthetic joint stabilization. Gene therapy is also a theoretically attractive technique to express BMPs clinically, since long-term, regulatable gene expression and systemic delivery with tissue-specific expression may be possible in future. This study was performed to determine whether an adenoviral vector containing the BMP-2 gene can be used to express BMP-2 in vitro and promote endochondral bone formation in vivo. In vitro, U87 MG cells transduced per cell with 20 MOI of an adenoviral construct containing the BMP-2 gene under the control of the universal CMV promoter (Ad-BMP-2) showed positive antibody staining for the BMP-2 protein at posttransfection day 2. The synthesis and secretion of active BMP-2 into the conditioned medium of Ad-BMP-2-transduced 293 cells were confirmed by Western blot analysis and the induction of alkaline phosphatase activity in a W-20 stromal cell assay. In vivo, Sprague-Dawley rats and athymic nude rats were injected with Ad-BMP-2 in the thigh musculature and were sacrificed on day 3, 6, 9, 12, 16, 21, 60, and 110 for histological analysis. The Sprague-Dawley rats showed evidence of acute inflammation, without ectopic bone formation, at the injection sites. In the athymic nude rats, BMP-2 gene therapy induced mesenchymal stem cell chemotaxis and proliferation, with subsequent differentiation to chondrocytes. The chondrocytes secreted a cartilaginous matrix, which then mineralized and was replaced by mature bone. This study demonstrates that a BMP-2 adenoviral vector can be utilized to produce BMP-2 by striated muscle cells in athymic nude rats, leading to endochondral bone formation. However, in immunocompetent animals the endochondral response is attenuated, secondary to the massive immune response elicited by the first-generation adenoviral construct.

Percutaneous spinal fusion using bone morphogenetic protein-2 gene therapy.

OBJECT: Gene therapy has many potential applications in neurosurgery. One application involves bone morphogenetic protein-2 (BMP-2), a low-molecular-weight glycoprotein that induces bone formation in vivo. Numerous studies have demonstrated that the BMP-2 protein can enhance spinal fusion. This study was undertaken to determine whether direct injection of an adenoviral construct containing the BMP-2 gene can be used for spinal fusion. METHODS: Twelve athymic nude rats were used in this study. Recombinant, replication-defective type 5 adenovirus with the cytomegalovirus (CMV) promoter and BMP-2 gene (Ad-BMP-2) was used. A second adenovirus constructed with the CMV promoter and beta-galactosidase (beta-gal) gene (Ad-beta-gal) was used as a control. In three groups (four rats each) 7.5 microl of virus (5x10(8) particles/microl) was injected percutaneously and paraspinally at the lumbosacral junction: Group 1 received Ad-BMP-2 bilaterally; Group 2 received Ad-BMP-2 on the right, Ad-beta-gal on the left; and Group 3 received Ad-beta-gal bilaterally. Computerized tomography (CT) scans of the lumbosacral spine were obtained at 3, 5, 8, and 12 weeks. At 12 weeks, the animals were killed and underwent histological inspection. Ectopic bone formation was observed both on three-dimensionally reconstructed CT scans and histological examination in all rats at sites treated with Ad-BMP-2. Histological analysis demonstrated bone at different stages of maturity adjacent to the spinous processes, laminae, and transverse processes. CONCLUSIONS: Results of this study clearly demonstrated that it is possible to produce in vivo endochondral bone formation by using direct adenoviral construct injection into the paraspinal musculature, which suggests that gene therapy may be useful for spinal fusion in the future.

Heterodimeric bone morphogenetic proteins show enhanced activity in vitro and in vivo.

The bone morphogenetic proteins (BMPs), a subgroup of the TGF-beta gene super-family, are dimeric molecules involved in the growth, differentiation and repair of a wide variety of tissues. Based on the observation that several of the BMPs co-purify when isolated from bovine bone and that a pattern of co-localization exists during mouse embryogenesis, we co-expressed various combinations of BMPs in Chinese hamster ovary cells to test for possible heterodimer formation and activity. Transient co-expression of BMP-2 with either BMP-5, BMP-6 or BMP-7, or BMP-4 transiently co-expressed with BMP-7, resulted in more BMP activity than expression of any single BMP. Stable cell lines were then made in order to purify and characterize co-expressed BMPs in more detail. Co-expression of BMP-2 with BMP-7 yielded heterodimeric BMP-2/7 with a specific activity about 20-fold higher than BMP homodimers in an in vitro alkaline phosphatase induction assay. These heterodimers were also 5- to 10-fold more potent than BMP-2 in inducing cartilage and bone in an in vivo assay. Similar results were obtained with BMP-2/6 heterodimer. These experiments demonstrate the increased potency of several BMP heterodimers relative to BMP homodimers and support the hypothesis that such heterodimeric forms are likely to have natural biological functions.

Expression and characterization of bone morphogenetic protein-2 in Chinese hamster ovary cells.

Bone is a dynamic tissue that responds to many factors including vitamin D, parathyroid hormone, estrogen, calcitonin, and bone morphogenetic proteins (BMPs). The ability to stimulate new bone growth would permit novel therapies for situations where bone mass has been lost due to accident or disease. Purified BMP-2, in conjunction with a suitable matrix, is sufficient to stimulate the synthesis of new bone (Wang et al., 1990). We have expressed recombinant human BMP-2 at high levels in Chinese hamster ovary cells using methotrexate-mediated gene amplification. Several forms of BMP-2 are secreted from CHO cells: (1) an amino-terminal propeptide of 40-45 kDa, (23) a mature active 30 kDa homodimer consisting of 18-22 kDa subunits, and (3) a small amount of uncleaved 60 kDa precursor protein. The mature, active protein is predominantly a 30 kDa homodimer consisting of subspecies of 18 and 22 kDa which differ by proteolytic processing at their amino termini. Mature BMP-2 and propeptide contain high mannose and complex N-linked oligosaccharides, respectively. The molar amount of secreted, processed propeptide is approximately 5-fold higher than mature BMP-2 in conditioned medium. BMP-2 associates with both the extracellular matrix and the surface of CHO cells, which may in part account for the unequal levels of extracellular propeptide and mature forms of the molecule in the conditioned medium. Recombinant BMP-2 can be expressed in sufficient quantities to assess its therapeutic potential for bone regeneration.

Recombinant human bone morphogenetic protein induces bone formation.

We have purified and characterized active recombinant human bone morphogenetic protein (BMP) 2A. Implantation of the recombinant protein in rats showed that a single BMP can induce bone formation in vivo. A dose-response and time-course study using the rat ectopic bone formation assay revealed that implantation of 0.5-115 micrograms of partially purified recombinant human BMP-2A resulted in cartilage by day 7 and bone formation by day 14. The time at which bone formation occurred was dependent on the amount of BMP-2A implanted; at high doses bone formation could be observed at 5 days. The cartilage- and bone-inductive activity of the recombinant BMP-2A is histologically indistinguishable from that of bone extracts. Thus, recombinant BMP-2A has therapeutic potential to promote de novo bone formation in humans.

Low levels of herpes simplex virus thymidine- thymidylate kinase are not limiting for sensitivity to certain antiviral drugs or for latency in a mouse model.

Herpes simplex virus mutant KG111 contains a nonsense mutation at codon 44 of the viral thymidine kinase (tk) gene and produces low amounts of a truncated tk polypeptide. We tested mutant KG111 and related viruses that specify varying amounts of similar truncated tk polypeptides for their sensitivities to antiviral nucleoside analogs at different temperatures using plaque reduction assays. The results of these assays showed that the nonsense mutation confers high resistance to bromovinyldeoxyuridine (BVdU) at any temperature and temperature-dependent resistance to acyclovir (ACV), buciclovir (BCV), ganciclovir (DHPG), and fluoroiodoarabinouracil (FIAU). Above relatively low threshold levels of tk that varied depending on the drug tested, viruses exhibited full sensitivity to ACV, BCV, DHPG, and FIAU at 34 degrees. Below these threshold levels, however, decreases in drug sensitivity were linear with decreases in tk levels, forming the basis of a pharmacological assay for tk gene expression. Studies of thymidine (TdR) anabolism in infected 143 tk-cells showed that when high TdR concentrations were added to the medium, KG111 directed thymidine monophosphate (TMP) formation at rates consonant with the amount of tk polypeptide produced by the mutant. When low concentrations to TdR were added to the medium, however, KG111 directed TMP formation at a rate similar to that directed by wild-type virus, indicating that the truncation of the tk polypeptide had little or no effect on tk activity at 34 degrees. Subsequent anabolism to thymidine diphosphate and thymidine triphosphate was reduced in KG111-infected cells, indicating a defect in TMP kinase activity that explains this mutant's resistance to BVdU. Despite the low levels of tk and TMP kinase activity expressed by KG111, this mutant established reactivatable latent infections as efficiently as wild-type virus in a mouse model.

Mutation within the herpes simplex virus DNA polymerase gene conferring resistance to (R)-9-(3,4-dihydroxybutyl)guanine.

Five herpes simplex virus mutants known or presumed to contain mutations in their DNA polymerase genes conferring resistance to acyclovir and arabinosyladenine also proved to exhibit some degree of resistance to (R)-9-(3,4-dihydroxybutyl)guanine (buciclovir). For one mutant, a buciclovir resistance mutation was mapped to a region of the viral DNA polymerase gene proposed to encode the deoxynucleoside 5'-triphosphate binding domain. These data implicate the viral polymerase as a target of buciclovir action that contributes to its antiviral selectivity.

A community-based dental program for older adults.

The planning and implementation of a community-based outreach program for older adults is described. Objectives of the program were to provide dental health education to older persons at their place of residence, to improve access to dental care for that population, and to increase the number of older adults treated at a dental facility administered by the Department of Community Dentistry, University of Michigan. Data collected during encounters with participants are reported to supplement the description of the program. In the first year, 98 older adults (mean age 71.3 years) participated in the outreach program which was directed by a dental hygienist. Of those persons whose initial encounter was with the outreach program, 47 percent eventually contacted the dental care facility and 36 percent completed treatment. Persons who elected to seek treatment average 3.9 encounters with the hygienist during the program; persons who did not seek treatment averaged 2.2 encounters. Strengths and weaknesses of the program are discussed.