Vibrational spectroscopic analysis of blood for diagnosis of infections and sepsis: a review of requirements for a rapid diagnostic test.

Infections and sepsis represent a growing global burden. There is a widespread clinical need for a rapid, high-throughput and sensitive technique for the diagnosis of infections and detection of invading pathogens and the presence of sepsis. Current diagnostic methods primarily consist of laboratory-based haematology, biochemistry and microbiology that are time consuming, labour- and resource-intensive, and prone to both false positive and false negative results. Current methods are insufficient for the increasing demands on healthcare systems, causing delays in diagnosis and initiation of treatment, due to the intrinsic time delay in sample preparation, measurement, and analysis. Vibrational spectroscopic techniques can overcome these limitations by providing a rapid, label-free and low-cost method for blood analysis, with limited sample preparation required, potentially revolutionising clinical diagnostics by producing actionable results that enable early diagnosis, leading to improved patient outcomes. This review will discuss the challenges associated with the diagnosis of infections and sepsis, primarily within the UK healthcare system. We will consider the clinical potential of spectroscopic point-of-care technologies to enable blood analysis in the primary-care setting.

Intraoperative cone-beam CT spatial priors for diffuse optical fluorescence tomography.

A hybrid system for intraoperative cone-beam CT (CBCT) imaging and continuous-wave fluorescence tomography (FT) has been developed using an image-guidance framework. Intraoperative CBCT images with sub-millimeter spatial resolution are acquired with a flat-panel C-Arm. Tetrahedral meshes are generated from CBCT for finite element method implementation of diffuse optical tomography (NIRFAST). Structural data from CBCT is incorporated directly into the optical reconstruction process using Laplacian-type regularization ('soft spatial priors'). Experiments were performed using an in-house optical system designed for indocyanine green (ICG) fluorescence. A dynamic non-contact geometry was achieved using a stereoscopic optical tracker for real-time localization of a laser diode and CCD camera. Source and detector positions were projected onto the boundary elements of the tissue mesh using algorithms for ray-triangle intersection and camera lens calibration. Simulation studies showed the capabilities of a soft-prior approach, even in the presence of segmentation uncertainties. Experiments with ICG targets embedded in liquid phantoms determined the improvements in the quantification of the fluorophore yield, with errors of 85% and <20% for no priors and spatial priors, respectively. Similar results were observed with the ICG target embedded in ex vivo porcine loin, with errors of 52% and 12%, respectively. A proof-of-principal animal study was performed in a VX2-tumor in vivo rabbit model using liposomal nanoparticles co-encapsulating contrast for CT (iohexol) and fluorescence (ICG) imaging. Fusion of CBCT and FT reconstructions demonstrated concurrent anatomical and functional delineations of contrast enhancement around the periphery of the buccal tumor. These developments motivate future clinical translation of the FT system into an ongoing CBCT-guided head and neck surgery trial.

Navigated non-contact fluorescence tomography.

A non-contact approach for diffuse optical tomography (DOT) has been developed for on-demand image updates using surgical navigation technology. A stereoscopic optical tracker provides real-time localization of reflective spheres mounted to a laser diode and near-infrared camera. Standard camera calibration is combined with tracking data to determine the intrinsic camera parameters (focal length, principal point and non-linear lens distortion) and the tracker-to-camera transform. Tracker-to-laser calibration is performed using images of laser beam intersection with a tracked calibration surface. Source and detector positions for a finite-element DOT implementation are projected onto the boundary elements of the tissue mesh by finding ray-triangle intersections. A multi-stage model converts camera counts to surface flux by accounting for lens aperture settings, fluorescence filter transmittance, photodetector quantum efficiency, photon energy, exposure time, readout offset and camera gain. The image-guidance framework was applied to an in-house optical tomography system configured for indocyanine green (ICG) fluorescence. Mean target registration errors for camera and laser calibration were less than 1 mm. Surface flux measurements of total reflectance and fluorescence in Intralipid-based fluorescence phantoms (0-2 µg ml-1) had mean errors of 3.1% and 4.4%, respectively, relative to diffusion theory predictions. Spatially-resolved reflectance measurements in a calibrated optical phantom agreed with theory for radial distances up to 25 mm from the laser source. Inverse fluorescence reconstructions of a sub-surface fluorescence target confirmed the localization accuracy (average target centroid error of 0.44 mm). This translational research system is under investigation for clinical applications in head and neck surgery, including oral cavity tumor resection, lymph node mapping and free-flap perforator assessment.

Matrix metalloproteinase-based photodynamic molecular beacons for targeted destruction of bone metastases in vivo.

The metastatic spread of cancer from the primary site or organ is one of its most devastating aspects, being responsible for up to 90% of cancer-associated mortality. Bone is one of the common sites of metastatic spread, including the vertebrae. Regardless of the treatment strategy, the clinical goals for patients with vertebral metastases are to improve the quality of life by preventing neurologic decline, to achieve durable pain relief and enhance local tumor control. However, in part due to the close proximity of the spinal cord, current treatment options are limited. We propose a novel therapeutic strategy with the use of photodynamic molecular beacons (PMBs) for targeted destruction of spinal metastases, particularly to de-bulk lesions as an adjuvant to vertebroplasty or kyphoplasty in order to mechanically stabilize weak or fractured vertebrae. The PDT efficacy of a matrix metalloproteinase-specific PMB is reported in a metstatic model that recapitulates the clinical features of tumor growth within the bone. We demonstrate that not only does tumor cell destruction occur but also the killing of bone stromal cells. The potential of PMB-PDT to destroy metastatic tumors, disrupt the osteolytic cycle and better preserve critical organs with an increased therapeutic window compared with conventional photosensitizers is demonstrated.

A spectrally constrained dual-band normalization technique for protoporphyrin IX quantification in fluorescence-guided surgery.

We report a dual-band normalization technique for in vivo quantification of the metabolic biomarker, protoporphyrin IX (PpIX), during brain tumor resection procedures. The accuracy of the approach was optimized in tissue simulating phantoms with varying absorption and scattering properties, validated with fluorimetric assessments on ex vivo brain tissue, and tested on human data acquired in vivo during fluorescence-guided surgery of brain tumors. The results demonstrate that the dual-band normalization technique allows PpIX concentrations to be accurately quantified by correction with reflectance data recorded and integrated within only two narrow wavelength intervals. The simplicity of the method lends itself to the enticing prospect that the method could be applicable to wide-field applications in quantitative fluorescence imaging and dosimetry in photodynamic therapy.

Suberoylanilide hydroxamic acid, a histone deacetylase inhibitor, protects dopaminergic neurons from neurotoxin-induced damage.

BACKGROUND AND PURPOSE: Prevention or disease-modifying therapies are critical for the treatment of neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease and Huntington's disease. However, no such intervention is currently available. Growing evidence has demonstrated that administration of histone deacetylase (HDAC) inhibitors ameliorates a wide range of neurologic and psychiatric disorders in experimental models. Suberoylanilide hydroxamic acid (SAHA) was the first HDAC inhibitor approved by the Food and Drug Administration for the sole use of cancer therapy. The purpose of this study was to explore the potential new indications of SAHA for therapy of neurodegenerative diseases in in vitro Parkinson's disease models. EXPERIMENTAL APPROACH: Mesencephalic neuron-glia cultures and reconstituted cultures were used to investigate neurotrophic and neuroprotective effects of SAHA. We measured toxicity in dopaminergic neurons, using dopamine uptake assay and morphological analysis and expression of neurotrophic substances by enzyme-linked immunosorbent assay and real-time RT PCR. KEY RESULTS: In mesencephalic neuron-glia cultures, SAHA displayed dose- and time-dependent prolongation of the survival and protection against neurotoxin-induced neuronal death of dopaminergic neurons. Mechanistic studies revealed that the neuroprotective effects of SAHA were mediated in part by promoting release of neurotrophic factors from astroglia through inhibition of histone deacetylation. CONCLUSION AND IMPLICATIONS: The novel neurotrophic and neuroprotective effects of SAHA demonstrated in this study suggest that further study of this HDAC inhibitor could provide a new therapeutic approach to the treatment of neurodegenerative diseases.

A 2D MEMS mirror with sidewall electrodes applied for confocal MACROscope imaging.

This paper presents microelectromechanical system micromirrors with sidewall electrodes applied for use as a Confocal MACROscope for biomedical imaging. The MACROscope is a fluorescence and brightfield confocal laser scanning microscope with a very large field of view. In this paper, a microelectromechanical system mirror with sidewall electrodes replaces the galvo-scanner and XYZ-stage to improve the confocal MACROscope design and obtain an image. Two micromirror-based optical configurations are developed and tested to optimize the optical design through scanning angle, field of view and numerical aperture improvement. Meanwhile, the scanning frequency and control waveform of the micromirror are tested. Analysing the scan frequency and waveform becomes a key factor to optimize the micromirror-based confocal MACROscope. When the micromirror is integrated into the MACROscope and works at 40 Hz, the micromirror with open-loop control possesses good repeatability, so that the synchronization among the scanner, XYZ-stage and image acquisition can be realized. A laser scanning microscope system based on the micromirror with 2 µm width torsion bars was built and a 2D image was obtained as well. This work forms the experimental basis for building a practical confocal MACROscope.

Local treatment of mixed osteolytic/osteoblastic spinal metastases: is photodynamic therapy effective?

The widespread use of systemic and local therapies aimed at spinal metastatic lesions secondary to breast cancer has increased the incidence of mixed osteolytic/osteoblastic patterns of bony disease. The complex structure of these lesions requires novel therapeutic approaches to both reduce tumor burden and restore structural stability. In photodynamic therapy (PDT), a minimally invasive approach can be used to employ light to activate a photosensitizing agent that preferentially accumulates in tumor tissue, leading to cell toxicity and death. Previous work in an osteolytic rat model (MT-1) demonstrated that PDT effectively ablates tumor and improves vertebral structural properties. The aim of this study was to assess the efficacy of PDT in a rat model of mixed osteolytic/osteoblastic spinal metastases. Mixed spinal metastases were generated through intracardiac injection of Ace-1 canine prostate cancer cells into female athymic rats (day 0). A single PDT treatment was applied to lumbar vertebra L2 of tumor-bearing and healthy control rats (day 14). PDT-treated and untreated control rats were euthanized and excised spines imaged with µCT to assess bone quality (day 21). Spines were mechanically tested or histologically processed to assess mechanical integrity, tumor burden, and remodelling properties. Untreated tumor-bearing vertebrae showed large areas of osteolysis and areas of immature, new bone formation. The overall bone quality resulting from these lesions consisted of decreased structural properties but without a significant reduction in mechanical integrity. PDT was shown to significantly decrease tumor burden and osteoclastic activity, thereby improving vertebral bone structural properties. While non-tumor-bearing vertebrae exhibited significantly more new bone formation following PDT, the already heightened level of new bone formation in the mixed tumor-bearing vertebrae was not further increased. As such, the effect of PDT on mixed metastases may be more influenced by suppression of osteoclastic resorption as opposed to the triggering of new bone formation.

Analytic expression of fluorescence ratio detection correlates with depth in multi-spectral sub-surface imaging.

Here we derived analytical solutions to diffuse light transport in biological tissue based on spectral deformation of diffused near-infrared measurements. These solutions provide a closed-form mathematical expression which predicts that the depth of a fluorescent molecule distribution is linearly related to the logarithm of the ratio of fluorescence at two different wavelengths. The slope and intercept values of the equation depend on the intrinsic values of absorption and reduced scattering of tissue. This linear behavior occurs if the following two conditions are satisfied: the depth is beyond a few millimeters and the tissue is relatively homogeneous. We present experimental measurements acquired with a broad-beam non-contact multi-spectral fluorescence imaging system using a hemoglobin-containing diffusive phantom. Preliminary results confirm that a significant correlation exists between the predicted depth of a distribution of protoporphyrin IX molecules and the measured ratio of fluorescence at two different wavelengths. These results suggest that depth assessment of fluorescence contrast can be achieved in fluorescence-guided surgery to allow improved intra-operative delineation of tumor margins.

Androgens induce oxidative stress and radiation resistance in prostate cancer cells though NADPH oxidase.

Androgen deprivation therapy (ADT) facilitates the response of prostate cancer (PC) to radiation. Androgens have been shown to induce elevated basal levels of reactive oxygen species (ROS) in PC, leading to adaptation to radiation-induced cytotoxic oxidative stress. Here, we show that androgens increase the expression of p22(phox) and gp91(phox) subunits of NADPH oxidase (NOX) and ROS production by NOX2 and NOX4 in PC. Pre-radiation treatment of 22Rv1 human PC cells with NOX inhibitors sensitize the cells to radiation similarly to ADT, suggesting that their future usage may spare the need for adjuvant ADT in PC patients undergoing radiation.

Image guided photothermal focal therapy for localized prostate cancer: phase I trial.

PURPOSE: We ascertained the feasibility and safety of image guided targeted photothermal focal therapy for localized prostate cancer. MATERIALS AND METHODS: Twelve patients with biopsy proven low risk prostate cancer underwent interstitial photothermal ablation of the cancer. The area of interest was confirmed and targeted using magnetic resonance imaging. Three-dimensional ultrasound was used to guide a laser to the magnetic resonance to ultrasound fused area of interest. Target ablation was monitored using thermal sensors and real-time Definity contrast enhanced ultrasound. Followup was performed with a combination of magnetic resonance imaging and prostate biopsy. Validated quality of life questionnaires were used to assess the effect on voiding symptoms and erectile function, and adverse events were solicited and recorded. RESULTS: Interstitial photothermal focal therapy was technically feasible to perform. Of the patients 75% were discharged home free from catheter the same day with the remainder discharged home the following day. The treatment created an identifiable hypovascular defect which coincided with the targeted prostatic lesion. There were no perioperative complications and minimal morbidity. All patients who were potent before the procedure maintained potency after the procedure. Continence levels were not compromised. Based on multicore total prostate biopsy at 6 months 67% of patients were free of tumor in the targeted area and 50% were free of disease. CONCLUSIONS: Image guided focal photothermal ablation of low risk and low volume prostate cancer is feasible. Early clinical, histological and magnetic resonance imaging responses suggest that the targeted region can be ablated with minimal adverse effects. It may represent an alternate treatment approach to observation or delayed standard therapy in carefully selected patients. Further trials are required to demonstrate the effectiveness of this treatment concept.

Extending immunofluorescence detection limits in whole paraffin-embedded formalin fixed tissues using hyperspectral confocal fluorescence imaging.

A major problem in microscopic imaging of ex vivo tissue sections stained with fluorescent agents (e.g. antibodies, peptides) is the confounding presence of background tissue autofluorescence. Autofluorescence limits (1) the accuracy of differentiating background signals from single and multiple fluorescence labels and (2) reliable quantification of fluorescent signals. Advanced techniques such as hyperspectral imaging and spectral unmixing can be applied to essentially remove this autofluorescent signal contribution, and this work attempts to quantify the effectiveness of autofluorescence spectral unmixing in a tumour xenograft model. Whole-specimen single-channel fluorescence images were acquired using excitation wavelengths of 488 nm (producing high autofluorescence) and 568 nm (producing negligible autofluorescence). These single-channel data sets are quantified against hyperspectral images acquired at 488 nm using a prototype whole-slide hyperspectral fluorescence scanner developed in our facility. The development and further refinement of this instrument will improve the quantification of weak fluorescent signals in fluorescence microscopy studies of ex vivo tissues in both preclinical and clinical applications.

Treatment of canine osseous tumors with photodynamic therapy: a pilot study.

Photodynamic therapy uses nonthermal coherent light delivered via fiber optic cable to locally activate a photosensitive chemotherapeutic agent that ablates tumor tissue. Owing to the limitations of light penetration, it is unknown whether photodynamic therapy can treat large osseous tumors. We determined whether photodynamic therapy can induce necrosis in large osseous tumors, and if so, to quantify the volume of treated tissue. In a pilot study we treated seven dogs with spontaneous osteosarcomas of the distal radius. Tumors were imaged with MRI before and 48 hours after treatment, and the volumes of hypointense regions were compared. The treated limbs were amputated immediately after imaging at 48 hours and sectioned corresponding to the MR axial images. We identified tumor necrosis histologically; the regions of necrosis corresponded anatomically to hypointense tissue on MRI. The mean volume of necrotic tissue seen on MRI after photodynamic therapy was 21,305 mm(3) compared with a pretreatment volume of 6108 mm(3). These pilot data suggest photodynamic therapy penetrates relatively large canine osseous tumors and may be a useful adjunct for treatment of bone tumors.

Efficacy assessment of sustained intraperitoneal paclitaxel therapy in a murine model of ovarian cancer using bioluminescent imaging.

We evaluated the pre-clinical efficacy of a novel intraperitoneal (i.p.) sustained-release paclitaxel formulation (PTX(ePC)) using bioluminescent imaging (BLI) in the treatment of ovarian cancer. Human ovarian carcinoma cells stably expressing the firefly luciferase gene (SKOV3(Luc)) were injected i.p. into SCID mice. Tumour growth was evaluated during sustained or intermittent courses of i.p. treatment with paclitaxel (PTX). In vitro bioluminescence strongly correlated with cell survival and cytotoxicity. Bioluminescent imaging detected tumours before their macroscopic appearance and strongly correlated with tumour weight and survival. As compared with intermittent therapy with Taxol, sustained PTX(ePC) therapy resulted in significant reduction of tumour proliferation, weight and BLI signal intensity, enhanced apoptosis and increased survival times. Our results demonstrate that BLI is a useful tool in the pre-clinical evaluation of therapeutic interventions for ovarian cancer. Moreover, these results provide evidence of enhanced therapeutic efficacy with the sustained PTX(ePC) implant system, which could potentially translate into successful clinical outcomes.

Optical molecular imaging: from single cell to patient.

Optical imaging is an emerging field with a wide range of biomedical research and clinical applications, both current and future. It comprises several classes of techniques that are capable of providing information at the molecular, cellular, tissue, and whole-animal levels. These techniques match well with emerging genomic and proteomic technologies that enable development of optical "probes," as well as with nanotechnologies for multifunctional imaging and drug delivery. These advances have enormous potential to accelerate drug discovery/development by providing predictive information on mechanisms of action and biological responses.

Transplantation of porous tubes following spinal cord transection improves hindlimb function in the rat.

STUDY DESIGN: Experimental. OBJECTIVE: To determine the effects of a porous tube transplant in spinal cord transected rats. SETTING: Acadia University, Wolfville, Nova Scotia, Canada. METHODS: Female rats were randomly assigned to three experimental groups: control (Con, n=8), spinal cord transected (Tx, n=5) and spinal cord transected with transplant (TxTp, n=7). The rats in the TxTp and Tx groups received a complete spinal cord transection at the T10 level and the TxTp group immediately received a porous tube transplant. RESULTS: Locomotor activity rated on the Basso, Beattie, Bresnahan scale improved significantly in the TxTp animals over the 4 weeks such that final scores were 21, 1.4 and 7.1 for the Con, Tx and TxTp groups, respectively. As expected, the muscle to body mass ratios of the hindlimb skeletal muscles of the Tx group were decreased (soleus 35%, plantaris 29% and gastrocnemius 29%) and this was also observed in the TxTp group (soleus 33%, plantaris 23% and gastrocnemius 30%). Cytochrome c oxidase (CYTOX) activity in the plantaris was decreased by Tx but maintained in the TxTp group (Con=82.2, Tx=44.8 and TxTp=72.8 U/min/g). CONCLUSION: Four weeks after the spinal cord transection, plantaris CYTOX activity and locomotor function improved with porous tube implantation. SPONSORSHIP: Natural Sciences and Engineering Research Council.

Vascular targeted photodynamic therapy with palladium-bacteriopheophorbide photosensitizer for recurrent prostate cancer following definitive radiation therapy: assessment of safety and treatment response.

PURPOSE: Tookad is a novel intravascular photosensitizer. When activated by 763 nm light, it destroys tumors by damaging their blood supply. It then clears rapidly from the circulatory system. To our knowledge we report the first application of Tookad vascular targeted photodynamic therapy in humans. We assessed the safety, pharmacokinetics and preliminary treatment response as a salvage procedure after external beam radiation therapy. MATERIALS AND METHODS: Patients received escalating drug doses of 0.1 to 2 mg/kg at a fixed light dose of 100 J/cm or escalated light doses of 230 and 360 J/cm at the 2 mg/kg dose. Four optical fibers were placed transperineally in the prostate, including 2 for light delivery and 2 for light dosimetry. Treatment response was assessed primarily by hypovascular lesion formation on contrast enhanced magnetic resonance imaging and transrectal ultrasound guided biopsies targeting areas of lesion formation and secondarily by serum prostate specific antigen changes. RESULTS: Tookad vascular targeted photodynamic therapy was technically feasible. The plasma drug concentration was negligible by 2 hours after infusion. In the drug escalation arm 3 of 6 patients responded, as seen on magnetic resonance imaging, including 1 at 1 mg/kg and 2 at 2 mg/kg. The light dose escalation demonstrated an increasing volume of effect with 2 of 3 patients in the first light escalation cohort responding and all 6 responding at the highest light dose with lesions encompassing up to 70% of the peripheral zone. There were no serious adverse events, and continence and potency were maintained. CONCLUSIONS: Tookad vascular targeted photodynamic therapy salvage therapy is safe and well tolerated. Lesion formation is strongly drug and light dose dependent. Early histological and magnetic resonance imaging responses highlight the clinical potential of Tookad vascular targeted photodynamic therapy to manage post-external beam radiation therapy recurrence.

In vivo quantification of fluorescent molecular markers in real-time by ratio imaging for diagnostic screening and image-guided surgery.

Future applications of "molecular diagnostic screening" and "molecular image-guided surgery" will demand images of molecular markers with high resolution and high throughput (~ > or =30 frames/second). MRI, SPECT, PET, optical fluorescence tomography, hyper-spectral fluorescence imaging, and bioluminescence imaging do not offer such high frame rates. 2D optical fluorescence imaging can provide surface images with high resolution and high throughput. The ability to accurately quantify the fluorescence in vivo is critical to extract functional information of the disease state, however few methods are available. Here, a ratiometric 2D quantification method is introduced. Through mathematical modeling the performance was evaluated using optical properties that resembled biological tissues with the fluorescent marker Protoporhyrin IX. Experimentally the performance was evaluated in optical phantoms with different optical properties employing a novel prototype clinical imaging system. The clinical feasibility of real-time, image-guided surgery was demonstrated in patients undergoing prostatectomy. Discussed are the reasons why the introduced method leads to an increased quantification performance followed by modifications so it can be applied to novel fluorescent molecular markers as phthalocyanine 4 and dual-fluorescent markers. These offer additional advantages as these can provide a linear response to marker concentration and further minimize the dependence on autofluorescence and optical properties, as demonstrated through modeling.

Increased expression of mitochondrial benzodiazepine receptors following low-level light treatment facilitates enhanced protoporphyrin IX production in glioma-derived cells in vitro.

BACKGROUND AND OBJECTIVES: This study investigates whether low-level light treatment (LLLT) can enhance the expression of peripheral-type mitochondrial benzodiazepine receptors (PBRs) on glioma-derived tumor cells, and by doing so promote the synthesis of protoporphyrin IX (PpIX) and increase the photodynamic therapy (PDT)-induced cell kill using 5-aminolevulinic acid (ALA). The endogenous photosensitizer, PpIX and related metabolites including coproporphyrin III are known to traffic into or out of the mitochondria via the PBRs situated on the outer mitochondrial membrane. Cells of astrocytic derivation within the brain express PBRs, while neurons express the central-type of benzodiazepine receptor. STUDY DESIGN: Astrocytoma-derived CNS-1 cells were exposed to a range of differing low-level light protocols immediately prior to PDT. LLLT involved using broad-spectrum red light of 600-800 nm or monochromatic laser light specific to 635 or 905 nm wavelength. Cells (5 x 10(5)) were exposed to a range of LLLT doses (0, 1, or 5 J/cm(2)) using a fixed intensity of 10 mW/cm(2) and subsequently harvested for cell viability, immunofluorescence, or Western blot analysis of PBR expression. The amount of PpIX within the cells was determined using chemical extraction techniques. RESULTS: Results confirm the induction of PBR following LLLT is dependent on the dose and wavelength of light used. Broad-spectrum red light provided the greatest cell kill following PDT, although LLLT with 635 nm or 905 nm also increased cell kill as compared to PDT alone. All LLLT regimens increased PBR expression compared to controls with corresponding increases in PpIX production. CONCLUSIONS: These data suggest that by selectively increasing PBR expression in tumor cells, LLLT facilitates enhanced tumor cell kill using ALA-PDT. This may further improve the selectivity and efficacy of PDT treatment of brain tumors.

Microtomographic analysis of healing of femtosecond laser bone calvarial wounds compared to mechanical instruments in mice with and without application of BMP-7.

BACKGROUND AND OBJECTIVE: This study investigated the healing of femtosecond laser created wounds in an animal model. STUDY DESIGN: We have assessed the healing of critical size wounds in mice calvaria using three different wounding techniques: carbide bur, diamond end-cutting bur, and ultrafast femtosecond laser, and in the presence or absence of bone morphogenetic protein-7 (BMP). Wound closure was examined using microcomputerized tomography at 3, 6, 9, and 12 weeks. RESULTS: Results have shown partial closure at up to 12 weeks with all techniques that did not involve the use of BMP, with the least closure noted in the laser groups as suggested by two-dimensional radiographic analysis. Bone volume measurements appeared slightly lower for the laser than for the mechanical groups, however statistically significant differences were seen only at week 6. No significant differences in closure were noted for the different methods in the BMP treated groups. CONCLUSIONS: Femtosecond laser cutting demonstrated an unsurpassed precision when compared to mechanical instruments. The addition of BMP led to very rapid healing with complete closure seen as early as 3 weeks and overcomes any potential healing delays that may arise from laser tissue cutting.