Ridge augmentation following implantation of recombinant human bone morphogenetic protein-2 in the dog.

BACKGROUND: Recombinant human bone morphogenetic protein-2 (rhBMP-2) in an absorbable collagen sponge (ACS) carrier induces bone for reconstruction of skeletal defects. The rhBMP-2/ACS implant is prepared by administering a rhBMP-2 solution to dry ACS. Once prepared, rhBMP-2/ACS forms a moldable, cohesive, and adhesive implant. However, rhBMP-2/ACS does not have sufficient structural strength to withstand soft tissue compression at specific anatomic sites. To more fully understand the mechanisms that affect bone induction by rhBMP-2/ACS in the presence of soft tissue compression, it would be useful to have a preclinical model that appropriately simulates such circumstances in patients. This pilot study evaluated one such potential model. METHODS: Bilateral, Class III alveolar defects were surgically produced in 4 adult mongrel dogs following extraction of the mandibular fourth premolars and reduction of the alveolar ridge. After an 8-week healing interval, mucoperiosteal flaps were elevated and rhBMP-2/ACS or rhBMP-2/ACS combined with hydroxyapatite (HA) was implanted into contralateral defects. The animals were euthanized at 12 weeks post-augmentation and block biopsies processed for histologic evaluation. RESULTS: Limited augmentation followed implantation of rhBMP-2/ACS (0.7 +/- 0.6 mm). In contrast, sites receiving rhBMP-2/ACS/HA exhibited clinically relevant ridge augmentation (5.5 +/- 1.6 mm). Defects implanted with rhBMP-2/ACS exhibited dense trabeculation within the corpus of the reduced alveolar process. The cortices appeared intact without evidence of expansion into the defect area. Three defects receiving rhBMP-2/ACS/HA exhibited sparse bone trabeculae amidst HA particles, fibrovascular tissue, and marrow. Commonly, the HA particles were encapsulated by fibrous tissue. Some particles were observed in contact with bone. CONCLUSIONS: The results suggests that rhBMP-2/ACS has limited effect alone in this augmentation model of Class III alveolar ridge defects. Inclusion of HA into the rhBMP-2 construct results in clinically relevant augmentation, however, the quality of bone is compromised.

Clinical evaluation of rhBMP-2/ACS in orthopedic trauma: a progress report.

Recombinant human bone morphogenetic protein-2 (rhBMP-2) is an osteoinductive protein that plays a pivotal role in bone growth and regeneration. Studies conducted in many mammalian species and, most recently, human clinical studies, have provided definitive evidence of the ability of rhBMP-2 to induce bone. The findings of these studies provide a rationale for investigating whether rhBMP-2 therapy will be clinically useful in orthopedic practice. Consequently, we have initiated a clinical research program to evaluate the safety and efficacy of rhBMP-2 therapy in open tibial fractures. A prospective observational study (involving no rhBMP-2 treatment) was conducted to define an appropriate patient population for rhBMP-2 clinical trials and has provided critical information related to the design and execution of future studies. The rhBMP-2 clinical trials include a small pilot feasibility study and several larger studies. Data from the pilot feasibility study have demonstrated that implantation of rhBMP-2 (combined with an absorbable collagen sponge) is surgically feasible and safe. Larger, multicenter clinical studies are now ongoing in the United States and abroad.

High level expression on a chimeric anti-ganglioside GD2 antibody: genomic kappa sequences improve expression in COS and CHO cells.

We report a flexible strategy for the high level expression of a recombinant human monoclonal antibody (mAb) in Chinese hamster ovary (CHO) cells, initially using COS monkey kidney cell transfections to evaluate rapidly modifications to immunoglobulin (Ig) DNA constructs. Using sequential transfections with two amplifiable markers, we generated CHO cell lines and clones that secrete 80-110 micrograms/10(6) cells/24 hours of a mouse-human chimeric IgG1 kappa mAb. This cellular productivity is considerably greater than most murine hybridomas and transfected myelomas. Our data also demonstrate that genomic kappa sequences can improve mAb expression in COS and CHO cells. As a paradigm, we focused our expression studies on a human chimeric form of 3F8, a murine mAb that binds to ganglioside GD2 on neuroblastoma and melanoma tumor cells.

Denaturing gradient gel electrophoresis identifies genomic DNA polymorphism with high frequency in maize.

We have used denaturing gradient gel electrophoresis (DGGE) to identify genomic DNA polymorphism in maize (Zea mays L.). DGGE probes detect polymorphism in maize at a frequency comparable to the incidence of restriction fragment length polymorphism (RFLP). Probes identifying polymorphism were mapped to maize chromosome arms by utilizing DGGE and maize lines carrying B-A chromosomal translocations. The methods for library construction, probe screening, and genome analysis, described here for maize, can also be applied to the genomic analysis of other organisms.

Nitrogen fixation by Klebsiella pneumoniae is inhibited by certain multicopy hybrid nif plasmids.

In our studies of nif gene regulation, we have observed that certain hybrid nif plasmids drastically inhibit the expression of the chromosomal nif genes of Klebsiella pneumonia. Wild-type (Nif+) K. pneumoniae strains that acquire certain hybrid nif plasmids also acquire the Nif- phenotype; these strains lose 90 to 99% of all detectable nitrogen fixation activity and grow poorly (or not at all) on solid media with N2 as the sole nitrogen source. We describe experiments which defined this inhibition of the Nif+ phenotype by hybrid nif plasmids and identify and characterize four nif DNA regions associated with this inhibition. We show that plasmids carrying these nif regions could recombine with, but not complement, nif chromosomal mutations. Our results suggest that inhibition of the Nif+ phenotype will provide a useful bioassay for some of the factors that mediate nif gene expression.

Overlapping sequences of Klebsiella pneumoniae nifDNA cloned and characterized.

A HindIII (17.0 kb) and an EcoR1 restriction fragment (6.9 kb) of Klebsiella pneumoniae nif DNA were cloned on two small amplifiable plasmids, pCM1 and pSA30 respectively. These plasmids between them carry 14 of the 15 known Klebsiella nif genes. The operon for the three structural genes for nitrogenase, nifpHDK, is carried on pSA30: four and five of the remaining six operons are on pCRA37 and pCM1 respectively. All of the nif genes were assigned to endonuclease restriction fragments of DNA using the Southern blotting technique (Southern, 1975) with total DNA of nif insertion mutants and radioactive plasmid DNA which contained cloned nif DNA sequences. Their locations were consistent with the genetic map of nif genes. The estimated size of the nif gene cluster was 24 kb.

Physical map of chromosomal nitrogen fixation (nif) genes of Klebsiella pneumoniae.

We describe a method for the rapid determination of the physical location of mutations caused by insertion of transposable elements. We used this method to construct a detailed physical map of the nitrogen fixation (nif) gene cluster of Klebsiella pneumoniae and to correlate it with the genetic map. Total cellular DNA was isolated from individual strains, each carrying an insertion in 1 of 15 different nif genes. The DNA was digested with a restriction endonuclease, fractionated by agarose gel electrophoresis, denatured, and blotted onto nitrocellulose filter paper. The DNA on the filters was hybridized with (32)P-labeled DNA fragments derived from amplifiable plasmids carrying cloned nif DNA fragments from K. pneumoniae. Altered hybridization patterns caused by insertions into nif genes allowed us to map nif mutations with respect to the previously mapped cleavage sites for various restriction endonucleases. We have used the same method to map the end points of nif deletions. Using this procedure, we assigned physical locations on the K. pneumoniae chromosome to 86 nif insertion mutations and 13 nif deletion end points. This mapping procedure provides a convenient alternative to deletion mapping as a definitive method for mapping insertion mutations within a gene or for ordering genes within a gene cluster. This procedure will be especially useful for mapping mutations conferring phenotypes that are difficult to monitor and for mapping mutations in bacterial species in which techniques for conducting deletion mapping have not been devised.

Recombinant plasmid that carries part of the nitrogen fixation (nif) gene cluster of Klebsiella pneumoniae.

We have cloned fragments of the Klebsiella pneumoniae genome that carry part of the his operon and part of the nitrogen fixation (nif) gene cluster on the amplifiable plasmid pMB9. One particular plasmid, pCRA37, complements mutations in the hisD, nifB, and nifF loci. The physical map of pCRA37 as determined by restriction enzyme analysis correlates with the genetic map of the his-nif region as determined previously by phage P1-mediated cotransductional analysis.