Effect of serum nicotine level on posterior spinal fusion in an in vivo rabbit model.

BACKGROUND CONTEXT: Cigarette smoking has a deleterious effect on spinal fusion. Although some studies have implied that nicotine is primarily responsible for poor fusion outcomes, other studies suggest that nicotine may actually stimulate bone growth. Hence, there may be a dose-dependent effect of nicotine on posterior spinal fusion outcomes. PURPOSE: The purpose of this study was to determine if such a relationship could be shown in an in vivo rabbit model. STUDY DESIGN/SETTING: This is a prospective in vivo animal study. METHODS: Twenty-four adult male New Zealand white rabbits were randomly divided into four groups. All groups received a single-level posterolateral, intertransverse process fusion at L5-L6 with autologous iliac crest bone. One group served as controls and only underwent the spine fusion surgery. Three groups received 5.25-, 10.5-, and 21-mg nicotine patches, respectively, for 5 weeks. Serum nicotine levels were recorded for each group. All animals were euthanized 5 weeks postoperatively, and spinal fusions were evaluated radiographically, by manual palpation, and biomechanically. Statistical analysis evaluated the dose response effect of outcomes variables and nicotine dosage. This study was supported by a portion of a $100,000 grant from the Orthopaedic Research and Education Foundation. Author financial disclosures were completed in accordance with the journal's guidelines; there were no conflicts of interests disclosed that would have led to bias in this work. RESULTS: The average serum levels of nicotine from the different patches were 7.8±1.9 ng/mL for the 5.25-mg patch group; 99.7±17.7 ng/mL for the 10.5-mg patch group; and 149.1±24.6 ng/mL for the 21-mg patch group. The doses positively correlated with serum concentrations of nicotine (correlation coefficient=0.8410, p<.001). The 5.25-mg group provided the best fusion rate, trabeculation, and stiffness. On the basis of the palpation tests, the fusion rates were control (50%), 5.25 mg (80%), 10.5 mg (50%), and 21 mg (42.8%). Radiographic assessment of trabeculation and bone incorporation and biomechanical analysis of bending stiffness ratio were also greatest in the 5.25-mg group. Radiographic evaluation showed a significant (p=.0446) quadratic effect of nicotine dose on spinal fusion. CONCLUSIONS: The effects of nicotine on spinal fusion are complex, may be dose dependent, and may not always be detrimental. The uniformly negative effects of smoking reported in patients undergoing spinal fusion may possibly be attributed to the other components of cigarette smoke.

Altering surface characteristics of polypropylene mesh via sodium hydroxide treatment.

Incisional hernias represent a serious and common complication following laparotomy. The use of synthetic (e.g. polypropylene) meshes to aid repair of these hernias has considerably reduced recurrence rates. While polypropylene is biocompatible and has a long successful clinical history in treating hernias and preventing reherniation, this material may suffer some limitations, particularly in challenging patients at risk of wound failure due to, for example, an exaggerated inflammation reaction, delayed wound healing, and infection. Surface modification of the polypropylene mesh without sacrificing its mechanical properties, critical for hernia repair, represents one way to begin to address these clinical complications. Our hypothesis is treatment of a proprietary polypropylene mesh with sodium hydroxide (NaOH) will increase in vitro NIH/3T3 cell attachment, predictive of earlier and improved cell colonization and tissue integration of polypropylene materials. Our goal is to achieve this altered surface functionality via enhanced removal of chemicals/oils used during material synthesis without compromising the mechanical properties of the mesh. We found that NaOH treatment does not appear to compromise the mechanical strength of the material, despite roughly a 10% decrease in fiber diameter. The treatment increases in vitro NIH/3T3 cell attachment within the first 72 h and this effect is sustained up to 7 days in vitro. This research demonstrates that sodium hydroxide treatment is an efficient way to modify the surface of polypropylene hernia meshes without losing the mechanical integrity of the material. This simple procedure could also allow the attachment of a variety of biomolecules to the polypropylene mesh that may aid in reducing the complications associated with polypropylene meshes today.

Nicotine Increases Osteoblast Activity of Induced Bone Marrow Stromal Cells in a Dose-Dependent Manner: An in vitro Cell Culture Experiment.

Previous studies by our group showed that nicotine delivered via a transdermal nicotine patch significantly enhanced posterior spinal fusion rates in rabbits. Nicotine transdermal patches provide a steady serum level; there may be a dose-dependent effect of nicotine on posterior spinal fusion. In an in vitro cell culture model of rabbit bone marrow-derived osteoblast-like cells, cells were exposed to different concentrations of nicotine (0, 20, 40, 80 ng/mL and 10, 100, 250 µg/mL). Wells were stained with an alkaline phosphatase (ALP) staining kit to determine ALP enzyme activity. Cells were stained with Von Kossa for mineralization. A two-way analysis of variance (ANOVA) using dose and time as variables showed significant differences among groups; post hoc analysis showed that the 100-µg/mL dose of nicotine significantly enhanced ALP activity over controls. A one-way ANOVA using dose as the variable showed that the 100- and 250-µg/mL doses had significantly greater mineralization than controls. Dose-response analysis revealed a statistically significant effect of nicotine dose on ALP activity and Von Kossa activity. The effects of nicotine on spinal fusion may be dose-dependent and due to stimulation of osteoblastic activity. Nicotine may not be responsible for the inhibited bone healing observed in smokers.

Incorporation and immunogenicity of cleaned bovine bone in a sheep model.

This study was conducted to determine if a novel cleaning process could extract antigenic material from bovine bone thereby improving incorporation. Cleaned bovine xenograft, untreated bovine xenograft and sheep allograft were implanted into the tibia of mature sheep for 12 and 24 weeks. Inflammation, bone integration and immunological reactions were evaluated via standardized assays. Cleaned bovine bone dowels induced significantly lower inflammatory responses (p < 50.05) when compared to traditionally processed xenograft. Bone integration, measured by in situ biomechanics, was not different between cleaned bovine bone and allograft controls (p = 0.96). A transient antibody response was observed for non-treated xenografts although this response abated by 3 months.

A quantitative polymerase chain reaction assay for detecting and identifying fungal contamination in human allograft tissue.

To complement donor selection and tissue processing, rapid and reliable detection, discrimination, and quantification of fungal pathogens are extremely important for tissues destined to be implanted into humans. The current detection method for fungal pathogens, in particular, is difficult and time-consuming. Quantitative polymerase chain reaction (qPCR) technology is considered one of the most sensitive methods to detect low levels of DNA. Here a qPCR method is described that can detect clinically relevant, pathogenic fungal organisms. The assay allowed the quantification of fungal organisms within a tissue implant and provides a means to identify the contaminating species. The primers for the qPCR assay were designed to amplify a conserved region of the L2 region of the large ribosomal subunit (LSU) gene. This set of primers was able to detect fewer than 10 colony forming units from Aspergillus and Candida species in spiked samples. Clinical samples were also evaluated using this method and the data compared positively to the existing accepted 28-day fungal culture method for fungal detection. The qPCR method described herein significantly reduced the time required to identify fungal contamination in tissue implants.

Induction of CD-RAP mRNA during periosteal chondrogenesis.

Induction of chondrogenesis and maintenance of the chondrocyte phenotype are critical events for autologous periosteal transplantation, which is a viable approach for cartilage repair. Cartilage-derived retinoic acid-sensitive protein (CD-RAP) is a recently discovered protein that is mainly produced in cartilage. During development, CD-RAP expression starts at the beginning of chondrogenesis and continues throughout cartilage maturation. In order to investigate the involvement of CD-RAP during periosteal chondrogenesis we have determined the nucleotide sequence of the rabbit CD-RAP mRNA and utilized this information to evaluate the temporal and spatial expression pattern of CD-RAP at the mRNA level during chondrogenesis. When the periosteal explants were cultured under chondrogenic conditions, the expression of CD-RAP was induced, as shown by a 40-fold increase in CD-RAP mRNA between days 7 and 10. The temporal expression pattern of CD-RAP closely mimicked that of collagen type IIB mRNA. Also, the CD-RAP mRNA was localized to the matrix forming chondrocytes in the cambium layer of the periosteum by in situ hybridization as indicated by colocalization with collagen type II mRNA and positive safranin O staining. These data suggest a regulatory role of CD-RAP in periosteal chondrogenesis, which is potentially important for both cartilage repair and fracture healing via callus formation.