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Improved Al ograft Disinfection Technique Using GCIB Joseph Khoury, PhD1; Sean R Kirkpatrick1; Son Chau1; Raymond Cherian1; Cameron McCaa1,2; Richard C Svrluga1; Laurence JB Tarrant, PhD1 INNOVATIONS IN SURFACE PROCESSING
Exogenesis Corp. Billerica, MA 01821
2McCaa Group, Atlanta, GA 30307
Materials and Methods
Background: Currently, soft tissue al ografts are classified in two broad groups; aseptical y harvested or
Tensile Strength Studies end-sterilized by irradiation or chemicals. Surgeons perceive problems with both. Some surgeons see aseptical y harvested tissues as "not clean enough" with potential for disease transmission. Others Bacterial studies: Study 1: E. coli DH5-α competent bacteria were transformed
Bacterial Studies perceive the end-sterilized tissues to be potential y damaged by irradiation or chemicals.
with pUC19 plasmid DNA to introduce an ampicillin-resistance gene in order to assay Purpose/Objective: We have sought to process soft tissues by a technique cal ed GCIB, which utilizes high
exogenously added bacteria. As described in Abstract, cleanly harvested goat flexor energy ionized gas clusters comprised of large numbers of inert argon atoms to modify surfaces. This Condition for Tendon
method results in a modified surface without any additional residues. We tested GCIB's effectiveness as a tendons were gently cleaned and cut into 2 cm long sections. Flexor tendon segments novel tissue disinfection method aimed to reduce or eliminate bio-burden, while preserving the inherent were placed in LB broth without- or with- ampicillin to determine background bacterial Fresh, incubated in LB mechanical and biological characteristics of al ograft tissues.
load. Flexor tendons were also inoculated with 1x106 colony forming units (CFU) Amp- Materials/Methods: Cleanly harvested goat flexor tendons were placed in a mild cleansing solution
Fresh, incubated in LB+AMP resistant E. coli and either left as controls or treated by GCIB at 5x1014 Ar clusters / cm2 consisting of PBS with 1% v/v Triton X-100, 2.5g/L Na deoxycholate, and 1% v/v penicil in/streptomycin Amp resistant E. coli, in LB+AMP overnight at 4oC. To assay for endogenous bacteria, flexor tendons were cut into 2 cm long sections and and then placed in LB broth with Amp overnight. Next day, aliquots of broth were placed overnight in culture broth (n=3) without- (control group 1) or with- (control group 2) ampicil in measured on a spectrophotometer and absorbances at 595nm were recorded.
Amp res E. coli + GCIB in LB+AMP
(Amp, 50µg/ml). Additional tendon sections were inoculated with 1x106 CFU Amp-resistant E. coli bacteria Dilutions of the broth were also spread onto LB agar plates with Amp and grown and frozen prior to control (test group I) or GCIB-treatment at a dose of 5x1014 Ar clusters/cm2 (test group overnight. Next day, colonies of bacteria were counted.
II, n=3). Fol owing treatment, tendons were incubated overnight in 5 ml culture broth with Amp in a Study 2: Collagen sheets cut to 0.25" x 0.5" were used as surrogates for soft tissue
Table 1. Study 1 results show a significant reduction in background bacteria in control group 2 shaker-incubator at 37oC. The next day, 1ml of each culture was assayed for growth on a when tendons are placed in Amp containing broth (* p<0.01). When Amp resistant bacteria are spectrophotometer at 595nm wavelength and absorbance was recorded (A595). 50µl of culture was then allografts. E. coli (ATCC 8739) were grown according to manufactures instructions and added to tendons, they grow wel in LB with Amp (Test 1), when similar tendons are treated by placed on pre-warmed LB-Agar-Amp plates in a dilution series and placed at 37oC overnight. The next day, dilution series were used to determine CFU of bacteria on LB agar plates. Collagen individual plates were scanned and colonies were counted. The impact of GCIB treatment on sheets were cut as described and 6 pieces were inoculated at 2.6x107 CFU / sample for GCIB, there are no bacterial colonies that survive (Test 2, * p<0.001).
biomechanical properties of goat tendons was evaluated by tensile testing on 18 matched pairs from three 15 minutes. Al pieces were then frozen at -80oC for 30 minutes. The pieces were then goats comparing fresh-frozen to GCIB-treated tendons. Tendons were loaded at a rate of 10 mm/min until failure using an 858 MiniBionix II MTS machine. Ultimate load and elongation at failure were measured lyophilized in a bench top lyophilizing unit for 1 hour. Three pieces were left as control, Escherichia coli (ATCC 8739) 2.6 x 107 CFU / Sample directly and ultimate stress, ultimate strain and Young's modulus were calculated for each sample. To three additional pieces were treated on both sides using GCIB at 5x1014 dose. Al the determine the effect of GCIB on cel attachment capacity of the tissue, fresh and GCIB-treated pig medial pieces of collagen were placed in 0.5ml LB broth for 1 hour at 370C. 1µl of broth was Total Count – Recovered CFU / Sample
col ateral ligaments (n=6) were decel ularized and then their surface were seeded with porcine then used for serial dilutions at 1:1,000 and 1:1,000,000 in LB broth and a subsequent ligamentous fibroblasts at a concentration of 106 cel s per ligament. The ligaments were incubated for 19 50µl were spread on individual LB Agar plates. Plates were incubated at 37oC over days in DMEM + 10% FBS. Ligaments were formalin fixed and slices were subjected to histological analysis to determine cel ular on-growth. Statistical analysis was performed using the Paired t-Test.
night. The next morning, individual colonies were counted to determine CFU / Sample.
Results: The use of the cleansing solution did not inhibit endogenous bacterial growth in the tendons of
Study 3: A validation of study 1 and 2 was performed at WuXi AppTec in Marietta, GA.
control group 1, which showed an A595 reading of 0.432 with over 500,000 colonies formed on the plates Essentially similar to study 2, however a total of 9 pieces were used, 2 as in-house in dilutions; however, the use of ampicil in (control group 2) did eliminate virtual y al bacteria from controls, 2 as shipping controls, and 5 as processed samples. Collagen pieces were growing [A595 = 0.001 and 4 colonies on the plates (p<0.01)]. When bacteria are added exogenously to No significant differences are found between fresh-frozen control goat flexor tendons to GCIB the tendons, the bacteria did grow well in ampicil in containing culture (test group I, A595 = 0.225, > inoculated at 2.7x107 CFU / sample, 7 were shipped back to Exogenesis in MA and 2 processed tendons in any of the mechanical testing properties. Cross section area, p=0.56; 500,000 colonies). Exogenously added bacteria are completely inhibited by GCIB processing [test group II, were maintained at the facility in GA. 5 pieces were further processed as described Table 2. Study 2 results show a significant decrease in recovered CFU/sample in the GCIB A595 = 0.000, 0 colonies (p<0.001)]. There were no significant differences between fresh-frozen and GCIB- above and all the pieces were sent back for analysis.
Ultimate load, p=0.64; Ultimate elongation, p=0.52; Ultimate stress, p=0.53; Ultimate strain, treated col agen samples (4-6) as compared to controls (1-3, p<0.02).
treated tendons as measured by ultimate load and elongation, as well as ultimate stress and strain or p=0.75; Tangent modulus, p=0.16.
Young's modulus. Control ligaments revealed one cel layer in the cel ular on-growth study; however, GCIB-treated ligaments displayed cel s growing in a multi-layer fashion.
Tensile Strength studies: Goat flexor tendons were cleanly harvested, cut to 6cm Cell Attachment Studies GCIB as a disinfection technique
Conclusion: GCIB may be used to reduce or eliminate organisms in the superficial layers of soft tissue
segments, and frozen at -80oC. 18 matched pairs from three goats were used to al ografts, without compromising the inherent mechanical integrity or the ability of the tissue to support compare fresh-frozen to GCIB-treated tendons. Treated tendons were lyophilized for 16 ) 10000000
cell growth and adherence in vitro.
hours prior to GCIB treatment at 5x1014 Ar clusters/cm2. Following treatment, all samples were analyzed at Clemson University and the Medical University of South Carolina. Al tendon segments were rehydrated in saline for 1.5 hours at room temperature and clamped onto an 858 MiniBionix II MTS machine and were loaded at a Introduction to GCIB
rate of 10 mm/min until failure. Ultimate load and elongation at failure were measured  Unique energetic ion bombardment directly and the initial cross sectional diameter and length were used to calculate ultimate stress, ultimate strain and Young's mdoulus.
 Clusters can be formed from a number of gases, Argon is typical y used due to inert Cell Attachment studies: Porcine anterior cruciate ligaments were cleanly  Clusters that collide with surface have harvested and ligamentous fibroblasts were grown out using an explant method.
Graph 1. Study 3 validation results from an outside vendor show a 5 log reduction in bacterial H&E staining reveals that GCIB treated MCL (right) do not inhibit cel attachment on the surface capability to modify material to about 10nm Medial colateral ligaments were decellularized using the method of Woods and Gratzer CFU/sample by GCIB treatment as compared to controls (* p<0.0001).
as compared with control ligaments (left).
below the surface.
(1), cut to 1.5cm segments, and either left as controls or treated by GCIB as described.
 Surface Treatment using Gas Cluster Ion Beams Porcine ligamentous fibroblasts were then seeded onto the surface of treated and un- treated MCL and allowed to attach and proliferate for 19 days. Ligaments were fixed in Questions or Further Information
 Changes the surface properties without formalin and parafin embedded, sections were then H&E stained to visualize cells.
changing the surface chemistry of a GCIB is a novel method in which soft tissue allografts may be Joseph Khoury, Ph.D.
disinfected effectively without the use of harsh chemical washes or Where needed, paired T-test were performed and p<0.05 was considered Director, Cell and Moleuclar Biology  Very shal ow penetration – at atomic levels significant. Power analysis were also performed for the Tensile Strength studies.
Exogenesis Corp.
gamma-irradiation. The effect of GCIB does not negatively impact  Extremely localized process that only impacts the surface INNOVATIONS IN SURFACE PROCESSING
bio-mechanical properties or the ability of cells to attach and Bil erica, MA 01821 (1) Woods T and Gratzer PF. Biomaterials 26 (2005) 7339–7349.
Tel: (978) 439-0120 proliferate on the surface.


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