Femtosecond Plasma Mediated Laser Ablation Has Advantages Over Mechanical Osteotomy of Cranial Bone:Medical Fibers

Keywords:medical fibers, medical laser,  Time:26-02-2016

Osteotomies fashioned with oscillating saws and drills using carbide or diamond cutting burs are the standard of care. However, the friction from cutting and grinding can cause significant thermal and mechanical trauma to adjacent bone tissue, leading to cell death and delayed healing [3,4]. In addition, mechanical instruments may be difficult to control, making fine, intricate, nonlinear cuts problematic. While the aim is to leave behind a cut edge that is well-suited for early cell attachment, extracellular matrix deposition, and tissue regeneration, use of mechanical instruments may yield a micro-environment suboptimal for bone regeneration. Recently, endoscopic surgery with an ultrasonic bone cutter (Sonopet Omni1, Synergetics Inc., St. Charles,MO) has been shown to prevent some of the traditional complications of scarring and intraoperative hemorrhage [5]. However, while this technique minimizes injury to adjacent soft tissue, there is still a potential for collateral damage to cells bordering the osteotomy site [6,7]. Medical Lasers present another alternative for tissue cutting, but can cause osseous tissue damage through photothermal, photoacoustic, photochemical, or photomechanical effects. While carbon dioxide (CO2) lasers, or other gas or solidstate lasers, have been successful at reducing mechanical friction, they have also been shown to have detrimental effects on healing [8–12]. Lasers with mid-infrared wavelengths, between 2.9 and 10.6 mm, have been evaluated for ablation on skin, aorta, femur, dentin, alveolar bone, cortical bone, and the skull. Importantly, thermal damage still occurs as a consequence of the tissue heating by the laser radiation absorbed in the target tissue [13–17]. Laser ablation aimed at correction of craniosynostosis using neodymium:yttrium-aluminum-garnet (Nd:YAG) and CO2 lasers also resulted in reduction of the bone regeneration rates due to degenerative changes from thermal damage [18]. Of note, short pulse lasers, such as 10 microsecond CO2 were shown tro emove dentin with only8 mm of thermal damage [19]. In contrast to the linear absorption of the laser radiation by the tissue, plasma-mediated ablation occurs in the focus of the laser beam when ultrashort pulses of high energy are applied. High electric field in the focal area results in ionization of the molecules and formation of plasma, leading to local absorption of the laser radiation, which allows heating the tissue by several thousand degrees during a single pulse. Due to the extremely short interaction time, plasma-mediated laser ablation results in minimal heat diffusion, and consequently, minimal collateral thermal damage. Plasma-mediated laser ablation has already been applied to dermis, cornea, blood vessels, brain, dentin, and bone [20–30]. Previous studies have shown that thermal damage to tissue resulting from plasma-mediated laser ablation can be below a single cell size (<0.2–3.0 mm) [24,31–33]. Since thermal damage is believed to be the most harmful side effect of laser ablation, the purpose of the present study was to therefore compare bone regeneration in calvarial defects created by a femtosecond titanium:sapphire (Ti:Sapphire) laser and conventional drill osteotomies.

MATERIALS AND METHODS
Laser System

The Ti:Sapphire femtosecond laser system (Tsunami, Spectra Physics, Santa Clara, CA) used in this study was tuned to 800 nm wavelength. Pulse duration was 150 femtosecond and repetition rate was set at 1 kHz. The pulse energy was measured with an Ophir Laser Star meter and a PE10-S detector (Ophir Optronics Ltd., Jerusalem, Israel). The average pulse energy was determined by measuring the output power at 1 kHz over an integration period of several seconds. Laser osteotomies were performed with an average pulse energy of 280 mJ. The beam was steered with dielectric mirrors and passed through a 55 mm focal length lens to focus onto the cranial surface into a minimum spot size of 10 mm Medical Fibers, as calculated based on Gaussian beam transformations. The laser was scanned with a two-axis scanning mirror (OIM100 Series, Optics in Motion, Long Beach, CA). A 4-mm diameter circular defect was made with a scanning speed of 4 mm/second. The mirror rotated at one revolution per second while the anesthetized mouse was kept stationary on a platform and required one rotation to ablate the entire depth of the cranium. The Ti:Sapphire laser was coaligned with a helium–neon (HeNe) laser operating at 632.8 nm for visual targeting. Since the mouse was kept stationary, there was no need for dynamic adjustment of the beam position and focusing.

Surgical Procedures

All experiments were performed in accordance with the Stanford University Animal Care and Use Committee guidelines. fifty-six-day-old wild-type CD1 mice were purchased from Charles River Laboratories (Wilmington, MA). Mice were housed in a light- and temperature-controlled environment and were given food and water ad libitum. For all surgical procedures, the mice were anesthetized with 7.5 mg/kg ketamine, 0.24 mg/kg acepromazine, and 1.5 mg/kg xylazine through an intraperitoneal injection. Under a dissecting microscope (Zeiss, Wetzlar, Germany), the surgical site was cleaned with ethanol, and an incision was made just off the sagittal midline to expose the calvaria. Periosteal tissue was removed and 4-mm calvarial defects were made in nonsuture-associated right parietal bone (700 mm thick) with the laser (n ¼ 7) or with a motorized 200 Series rotary tool (Dremel; Robert Bosch Tool, Racine, WI) using a 4-mm diamond-coated core drill bit (n ¼ 7). To minimize thermal injury, all drill defects were performed with constant saline irrigation to minimize thermal injury. In one additional mouse, critical size calvarial defects were created bilaterally. The laser was used to create a 4-mm calvarial defect in the right parietal bone, and the trephine drill bit was used to create a 4-mm calvarial defect in the left parietal bone. Because the dura mater has been shown to be critical for calvarial reossification, a circular piece of expanded polytetrafluoroethylene (ePTFE) surgical membrane (Gore-Tex; W.L. Gore & Associates, Inc., Flagstaff, AZ) was placed into the defect to function as a barrier between the dura and the edges of the bone defect (fig. 1A) [34]. Incisions were closed using 6–0 Vicryl sutures (Ethicon, Cornelia, GA), and the mice were kept under a warm lamp to maintain their internal temperature until awakening following the anesthesia. No wound infections developed in the animals. Following surgery, mice were kept for up to 8 weeks for micro-CT analysis. At the end of the eighth week and following the last CT scan, the mice were euthanized for histological analysis.

Micro-CT Imaging Analysis

For micro-CT scans, the mice were anesthetized with isoflurane. Imaging was performed using a Siemens Inveon MicroPET/CT scanner (Siemens Medical Solutions, Inc., Malvern, PA). Using our scan protocol parameters, each image with a 100 mm resolution was acquired in a total scan time of 10 minutes.