Bone Mineralization and Spinal Fusion Evaluation of a Truss-based Interbody Fusion Device: Ovine Finite Element Analysis with Confirmatory In Vivo Outcomes.

STUDY DESIGN: Finite element analysis (FEA) and in vivo ovine spinal interbody fusion study. OBJECTIVE: To determine comparative load-induced strain amplitudes, bone mineralization and fusion outcomes associated with different diameter struts in a truss-based interbody fusion device. SUMMARY OF BACKGROUND DATA: Additive manufacturing technology has been employed to develop implants that actively participate in the fusion process. The truss device enables the optimal transfer of compressive and tensile stresses via the struts. Mechanobiologic principles postulate that strut diameter can be regulated to allow different magnitudes of strain distribution within the struts which may affect fusion rates. METHODS: Modeling of strain distributions as a function of strut diameter (0.75, 1.0, 1.25, and 1.5 mm) employed FEA that simulated physiologic loading conditions. A confirmatory in vivo ovine lumbar spinal interbody fusion study compared fusion scores and bone histomorphometric variables for cages with 0.75 and 1.5 mm strut diameters. Outcomes were compared at 3-, 6-, and 12-month follow-up intervals. RESULTS: FEA showed an inverse association between strut diameter and peak strain amplitude. Cages with 1.0, 1.25, and 1.5 mm struts had peak strain values that were 36%, 60%, and 73% lower than the 0.75 mm strut strain value. In vivo results showed the mean fusion score for the 0.75 mm diameter strut cage was significantly greater by 3-months versus the 1.5 mm strut cage, and remained significantly higher at each subsequent interval (P < 0.001 for all comparisons). Fusion rates were 95%, 100%, and 100% (0.75 mm) and 72.7%, 86.4%, and 95.8% (1.5 mm) at 3, 6, and 12 months. Thinner struts had greater mineralized bone tissue and less fibrous/chondral tissue than the thicker struts at each follow-up. CONCLUSION: Validating FEA estimates, cages with smaller diameter struts exhibited more rapid fusion consolidation and more aggressive osseointegration compared with cages with larger diameters struts.Level of Evidence: 4.

Fortifying the Bone-Implant Interface Part 2: An In Vivo Evaluation of 3D-Printed and TPS-Coated Triangular Implants.

BACKGROUND: Minimally invasive surgical fusion of the sacroiliac (SI) joint using machined solid triangular titanium plasma spray (TPS) coated implants has demonstrated positive clinical outcomes in SI joint pain patients. Additive manufactured (AM), i.e. 3D-printed, fenestrated triangular titanium implants with porous surfaces and bioactive agents, such as nanocrystalline hydroxyapatite (HA) or autograft, may further optimize bony fixation and subsequent biomechanical stability. METHODS: A bilateral ovine distal femoral defect model was used to evaluate the cancellous bone-implant interfaces of TPS-coated and AM implants. Four implant groups (n=6/group/time-point) were included: 1)TPS-coated, 2)AM, 3)AM+HA, and 4)AM+Autograft. The bone-implant interfaces of 6- and 12-week specimens were investigated via radiographic, biomechanical, and histomorphometric methods. RESULTS: Imaging showed peri-implant bone formation around all implants. Push-out testing demonstrated forces greater than 2500 N, with no significant differences among groups. While TPS implants failed primarily at the bone-implant interface, AM groups failed within bone ~2-3mm away from implant surfaces. All implants exhibited bone ongrowth, with no significant differences among groups. AM implants had significantly more bone ingrowth into their porous surfaces than TPS-coated implants (p<0.0001). Of the three AM groups, AM+Auto implants had the greatest bone ingrowth into the porous surface and through their core (p<0.002). CONCLUSIONS: Both TPS and AM implants exhibited substantial bone ongrowth and ingrowth, with additional bone through growth into the AM implants' core. Overall, AM implants experienced significantly more bone infiltration compared to TPS implants. While HA-coating did not further enhance results, the addition of autograft fostered greater osteointegration for AM implants. CLINICAL RELEVANCE: Additive manufactured implants with a porous surface provide a highly interconnected porous surface that has comparatively greater surface area for bony integration. Results suggest this may prove advantageous toward promoting enhanced biomechanical stability compared to TPS-coated implants for SI joint fusion procedures.

Evaluation of a plasmid DNA-based anthrax vaccine in rabbits, nonhuman primates and healthy adults.

VCL-AB01, a cationic lipid-formulated plasmid DNA (pDNA)-based vaccine that contains genes encoding genetically detoxified Bacillus anthracis protective antigen (PA) and lethal factor (LF), was assessed in a Phase 1, dose-escalating clinical trial in healthy adults for safety and immunogenicity, and in nonhuman primates for immunogenicity and efficacy against challenge with a lethal dose of B. anthracis spores. Healthy 18-45 year old subjects were randomly assigned to receive either the investigational vaccine containing 0.2 mg, 0.6 mg, or 2 mg of total pDNA per dose, or saline placebo, administered at 0, 1 and 2 months. The 0.2 mg and 0.6 mg dose levels were generally well tolerated; however, dose-limiting reactogenicity was observed among subjects given the first 2 mg dose and the remaining two injections in the 2 mg group were reduced to 0.6 mg. Dose-related increases in seroconversion frequencies were observed. Overall, 10%, 33.3% and 80% of subjects in the 0.2, 0.6 and 2 mg groups, respectively, developed antibodies to PA and/or LF as measured by ELISA; however, antibodies with toxin neutralizing activity (TNA) were detected in only one subject. In monkeys that received a 0.6 mg dose three times at 2 week intervals, low levels of antibodies were detected by ELISA but not by the TNA assay in all animals just prior to challenge. Despite the absence of TNA, 75% animals survived the lethal challenge. In summary, VCL-AB01 was generally well tolerated in humans at a dose that provided immunity in monkeys despite the lack of robust TNA titers in either species.

Plasmid DNA-based vaccines protect mice and ferrets against lethal challenge with A/Vietnam/1203/04 (H5N1) influenza virus.

Plasmid DNA (pDNA) vaccines represent an alternative to conventional inactivated influenza vaccines that are likely to experience supply constraints during a pandemic. Several Vaxfectin-formulated pDNA vaccines were tested in mice and ferrets for efficacy against a lethal challenge with the highly pathogenic A/Vietnam/1203/04 (H5N1) influenza virus strain; the vaccines encoded influenza A virus hemagglutinin (HA), and/or nucleoprotein (NP), and M2 protein. Complete protection from death and disease was achieved in mice and ferrets with 2 doses of a Vaxfectin-formulated vaccine containing H5 HA, NP, and M2 plasmids and in ferrets with only 1 dose. A Vaxfectin-formulated vaccine containing NP and M2 pDNA provided significant protection against death in mice and provided some benefit in ferrets (i.e., 17% survival, delayed time to illness and death, and significant reduction in viral load compared with that in negative control animals). These experiments support the clinical testing of pDNA vaccine candidates that may ultimately increase global vaccine supply options during pandemics.

A TaqMan reverse transcription polymerase chain reaction (RT-PCR) in vitro potency assay for plasmid-based vaccine products.

A TaqMan-based reverse transcription polymerase chain reaction (RT-PCR) assay has been developed as an in vitro potency assay to measure the most immediate biological activity of plasmid DNA (pDNA)-based products. The assay measures transgene-specific messenger RNA (mRNA) from cultured cells transfected with VCL-CB01, a bivalent pDNA-based human cytomegalovirus (CMV) vaccine. The forward and reverse primers have been designed to make the RT-PCR reaction selective for plasmid-derived mRNA and to allow discrimination of expression levels of individual plasmids in a multivalent pDNA vaccine. The relative potency of a vaccine lot is assessed by transfecting reference and test samples into cultured cells in parallel and analyzing total RNA from the cells by RT-PCR. Statistical analysis of dose response data from reference material supports a parallel-line model for calculating relative potency. Preliminary data demonstrate the ability of this assay to distinguish product potencies at 50, 75, 150, and 200% of the reference material. In addition, forced degradation of pDNA demonstrates that a decrease in relative potency as measured by the RT-PCR assay in vitro correlates well with a decrease in CMV DNA vaccine-mediated humoral immune responses in mice injected with the same material.

IL-2 plasmid electroporation: from preclinical studies to phase I clinical trial.

Electroporation (EP)-assisted intralesional delivery of Interleukin-2 (IL-2) plasmid (pDNA) has the potential to increase the local concentration of the expressed cytokine for an extended time in the injected tumors while minimizing its systemic concentration, in comparison with systemic delivery of the recombinant cytokine. Nonclinical Investigational New Drug application-enabling studies were performed in mice to evaluate the effect of intratumoral administration of murine IL-2 pDNA on local expression and systemic distribution of IL-2 transgene as well as the inhibition of established tumor growth. The safety of repeated administrations of a human IL-2 pDNA product candidate with EP was evaluated in rats. Following the nonclinical safety and efficacy studies, a human IL-2 pDNA product candidate intralesionally administered with EP to metastatic melanoma patients is currently being investigated in a phase I clinical trial.

Vaccination with polymerase chain reaction-generated linear expression cassettes protects mice against lethal influenza A challenge.

The feasibility of a linear expression cassette (LEC)-based influenza A DNA vaccine was demonstrated in mice, using a lethal dose (LD90) of a mouse-adapted A/Hong Kong/8/68 (H3N2) influenza strain. LECs expressing hemagglutinin (HA) from either the homotypic H3N2 or the heterotypic H1N1 (A/Puerto Rico/8/34) influenza virus were produced by polymerase chain reaction and either phosphodiester- or phosphorothioate-modified oligonucleotide primers. Survival subsequent to lethal viral challenge was used as a primary end point; weight loss was the secondary end point. Survival and weight loss data showed that protection can be achieved in mice with 50 microg of phosphate-buffered saline-formulated LEC DNA or 2 microg of Vaxfectin-formulated LEC DNA. Survival correlated with neutralizing antibody titers (hemagglutination inhibition, HAI); titers obtained after vaccination with LEC were equivalent to those obtained with HA (H3N2) plasmid DNA control. Vaccination with heterotypic H1 HA-LEC DNA provided no protection against viral challenge.

Vaxfectin-formulated influenza DNA vaccines encoding NP and M2 viral proteins protect mice against lethal viral challenge.

Next generation influenza vaccines containing conserved antigens may enhance immunity against seasonal or pandemic influenza virus strains. Using a plasmid DNA (pDNA)-based vaccine approach, we systematically tested combinations of NP, M1, and M2 antigens derived from consensus sequences for protection against lethal influenza challenge and compared formulations for adjuvanting low pDNA vaccine doses. The highest level of protection at the lowest pDNA doses was provided by Vaxfectin-formulated NP + M2. Vaxfectin adjuvanticity was confirmed with a low dose of HA pDNA. These promising proof-of-concept data support the clinical development of Vaxfectin-formulated pDNA encoding NP + M2 consensus proteins.

Comparison of rabbit and mouse models for persistence analysis of plasmid-based vaccines.

Experiments were conducted with a cationic lipid-formulated pDNA vaccine (VCL-AB01) to evaluate the models used to determine biodistribution, persistence and the potential for integration (into genomic DNA) of plasmid DNA-based vaccines. Mice were injected with a high-dose volume of 50 microL unilaterally containing approximately 1.33 x 10(13) plasmid copy numbers (PCN) or a low-dose volume of 20 microL bilaterally ( approximately 5.3 x 10(12) PCN). Rabbits were injected bilaterally with a 0.5 mL ( approximately 1.33 x 10(14) PCN) volume. Injection site muscle tissue was harvested two days, one month, and two months postinjection for the low-dose murine and rabbit models and two days and two months postinjection for the high-dose murine model. Total DNA was extracted and analyzed by real-time quantitative PCR for sequences specific to the injected pDNA. The geometric mean PCN/microg of total DNA from the high and low dose models were compared to determine if injection volume impacts clearance and/or persistence. Results from these studies showed that PCN clearance over two months was similar in mice injected with 20 microL and rabbits injected with 0.5 mL, but PCN clearance was slower in mice injected with similar PCN in 50 microL (1.33 x 10(13) PCN) compared to 20 microL (5.3 x 10(12) PCN). Persistence at two months in the rabbit and low-dose murine models was comparable, with geometric mean of 5.22 x 10(3) PCN/microg of total DNA for the low-dose volume murine model and 2.81 x 10(3)/microg DNA for the rabbit model. Interanimal variability in persistence was not impacted by dose volume.

I. Poloxamer-formulated plasmid DNA-based human cytomegalovirus vaccine: evaluation of plasmid DNA biodistribution/persistence and integration.

Preclinical studies were conducted in mice and rabbits to evaluate biodistribution/persistence and potential integration of plasmid DNA (pDNA) after intramuscular administration of a poloxamer-formulated pDNAbased vaccine, VCL-CT01, encoding gB, pp65, and IE1 human cytomegalovirus (hCMV) immunogens. Tissue distribution in mice vaccinated with VCL-CT01 was compared with that in mice vaccinated with a phosphate- buffered saline (PBS)-formulated control pDNA vaccine. Residual pDNA copy number (PCN), in selected tissues collected on days 3, 30, and 60 after vaccination, was measured by quantitative polymerase chain reaction. In VCL-CT01-vaccinated mice and in control pDNA-vaccinated mice, pDNA was below the limit of detection by day 60 in all tissues except the injection site. Clearance of pDNA from the injection site was slower in VCL-CT01-vaccinated mice compared with PBS-pDNA-vaccinated mice. An integration study was conducted in rabbits to determine whether pDNA integration into the genome of the vaccinated animal contributed to pDNA persistence. Residual pDNA in VCL-CT01-injected rabbit muscle collected 60 days after vaccination (geometric mean of 1085 PCN/microg total DNA) was comparable to that observed in VCL-CT01- injected mouse muscle (geometric mean of 1471 PCN/microg total DNA) collected at the same time point. pDNA integration was not detectable by column agarose gel electrophoresis despite the persistence of pDNA at the injection site 60 days after vaccination. Therefore the risk of genomic integration of hCMV pDNA formulated with poloxamer was considered negligible.

II. Cationic lipid-formulated plasmid DNA-based Bacillus anthracis vaccine: evaluation of plasmid DNA persistence and integration potential.

Several formulated plasmid DNA (pDNA)-based vaccines are being evaluated for safety and efficacy in healthy human subjects. A safety concern for any vaccine that contains genetic material, be it whole organism, live-attenuated, or gene-based, is the potential for integration into genomic DNA (gDNA). To address this concern, a preclinical pDNA persistence/integration study was conducted in rabbits to determine the level of pDNA in muscle 2, 28, and 64 days after intramuscular injection of DMRIE:DOPE-formulated pDNAs encoding Bacillus anthracis detoxified LF and PA proteins (VCL-AB01 vaccine). Total DNA was extracted from day 64 muscle tissue and fractionated by column agarose gel electrophoresis (CAGE). Plasmid copy number (PCN) in muscle 64 days after injection (geometric mean, 2808 PCN/microg of total DNA or 150,000 diploid genomes) was determined by quantitative polymerase chain reaction. Analysis of total DNA from five VCLAB01- injected rabbits revealed that two of five samples had no detectable PCN in the high molecular weight fraction after one round of CAGE, two samples had PCN under the lower limit of quantitation, and the remaining sample had 123 PCN/microg. All PCN in the latter sample cleared after an additional round of CAGE. It appears, therefore, that persisting PCN fractionate as low molecular weight material and are most likely not integrated into gDNA. Even if the worst-case assumption is made that the highest PCN found associated with gDNA represented covalently integrated pDNA inserts, the frequency of mutation would still be 500-fold lower than the autosomal spontaneous mutation rate.

Alveolar ridge augmentation using titanium micromesh: an experimental study in dogs.

PURPOSE: Augmentation of the alveolar ridge before implant placement is frequently performed. The purpose of this study was to compare the amount and quality of bone formation under Micro Titanium Augmentation Mesh (M-TAM) when used alone for guided tissue regeneration or in combination with a porous hydroxyapatite (HA) bone graft substitute. MATERIALS AND METHODS: Nine adult female dogs underwent extraction of premolars and molars and had a knife-edge ridge created. Three months later, the ridges were augmented with either M-TAM alone or M-TAM with a nonresorbable porous HA (Interpore 200; Interpore Cross International, Irvine, CA). Six months after augmentation, the dogs were killed, and the mandibles were harvested and imaged using 3-dimensional computed axial tomography. Statistical analysis was performed from the data obtained from the scans. The mandibles were then fixed, sectioned, and examined by light microscopy. RESULTS: Dehiscence occurred in 22 of 32 experimental sites. Seven of these 22 dehisced sites showed increased ridge width. Ridge width increased in both the HA and non-HA groups. The HA group showed a greater increase in ridge width. CONCLUSION: A high rate of dehiscence was observed in this animal study using M-TAM for guided tissue regeneration. In animals that did not dehisce, increased width was greater when nonresorbable porous HA was used as a bone graft substitute.

Osteoinduction by a collagen mineral composite combined with isologous bone marrow in a subcutaneous rat model.

In a subcutaneous implant rat model, Collagraft alone (n=8), Collagraft plus isologous bone marrow (n=8), and marrow alone (n=8) were evaluated. Twelve rats were euthanized at 11 days and 12 at 21 days. Explants were evaluated histologically for evidence of bone formation, and osteogenic activity was determined by an assay for alkaline phosphatase. Histological bone formation was absent in all groups at 11 days. At 21 days, in the Collagraft alone and bone marrow alone groups, no bone induction was noted. In contrast, 21-day specimens from the Collagraft plus bone marrow group showed newly formed bone. Alkaline phosphatase activity was negligible (<0.05 units/mg) in all 11-day specimens. Activity in Collagraft plus bone marrow specimens at 21 days (0.38 units/mg) was significantly higher than any other group (P<.001 for all comparisons). This study demonstrates that Collagraft plus bone marrow is an osteoinductive matrix.