0461 - Customization of Osteochondral Grafts and Graft Beds using a Novel Ultrafast Laser System

Customization of Osteochondral Grafts and Graft Beds using a Novel Ultrafast Laser SystemMelissa Prickaerts1,2, Elena Dybner1, Cameron Romanet1, Tom Dzelzainis3, Sabrina Hammouti3, Ahmad Golaraei3,4,5, Faiyza Alam3, Valentin Demidov2,4,I. Alex Vitkin2,4,6, Virginijus Barzda3,5, Lothar Lilge2,4, Robin Marjoribanks3, Margarete Akens1,2,71Techna Institute, University Health Network, 2Department of Medical Biophysics, University of Toronto, 3Department of Physics, University of Toronto, 4Princess Margaret Cancer Centre, University Health Network, 5Department of Chemical and Physical Sciences, University of Toronto Mississauga6Department of Radiation Oncology, University of Toronto, 7Department of Surgery, University of Toronto, Toronto, ON, CanadaDisclosures: Authors have no conflicts of interest to discloseINTRODUCTION: Treatment options for osteoarthritis vary depending on the disease stage, anything from local injections to total joint replacement1. Osteochondral grafts are used to address localized articular cartilage damage, preventing or delaying total joint replacement2. However, major grafting constraints include the damage incurred to tissues during preparation and the limited shapes and size of grafts available. Grafts are harvested using mechanical tools producing rounded grafts and frequently require mosaic-like placement in larger defects. Conventional long pulse and continuous pulse lasers cause substantial tissue damage to the cartilage and subchondral bone because of their thermal ablation mechanisms3. However, the use of an ultrafast burst-mode fibre laser that is suitable for arthroscopy would allow precise graft fitting by producing customized osteochondral grafts and recipient graft beds. The aim of this study is to determine optimal laser settings are by examine the effects of an ultrafast laser ablation on cartilage, evaluating the precision of cutting graft beds, and comparison outcomes of laser ablation to standard mechanical tools used in graft bed preparation.METHODS: Articular cartilage explants were harvested from heathy, euthanized Yorkshire pigs (previously used in non-musculoskeletal related, organ transplant experiments) from the distal femoral condyles and placed in phosphate buffered solution. The porcine cartilage explants underwent laser ablation with an ultrafast burst-mode ytterbium fibre laser system (FiberLAST Inc., 1037 nm wavelength, 350 fs pulse duration, 60 pulses per burst, 20 u03bcm spot) or mechanical graft bed preparation, the current standards for cutting cartilage and graft beds. Mechanical tools used were a surgical drill bit (straight shank with four flutes) and a 3 mm biopsy punch. To assess the damage as a result of laser ablation and mechanical manipulation, a live/dead fluorescence-based assay (Invitrogen) and confocal microscopy (Leica TCS SP8) were used. Analysis of confocal Z-stacks using Imaris software (Bitplane) produced surface regions identifying live and dead cells, which allowed for measuring cell death at the cutsu2019 margin. Scanning electron microscopy (SEM) (Hitachi SU3500 SEM) enabled the examination of the topography of cut faces. Histology sections of tissue were stained with hematoxylin and eosin (H&E) to reveal cellular structure and safranin-O to illustrate proteoglycan content. Polarization-in, polarization-out (PIPO) second harmonic generation (SHG) imaging of the H&E stained histology slides were used to assess the changes of collagen fibril organization within the extracellular matrix. Additionally, optical coherence tomography (OCT) provided information to perform the calculations of material removed during ablation. Using the measured cross-sectional area with the user-programmed laser scanning speeds provided material removal rates. Statistical analysis of the live/dead staining was performed using a 1-way ANOVA with a Bonferroni post-hoc test. Differences of p < 0.05 were considered statistical significant.RESULTS: Cartilage tissue was successfully ablated in predetermined geometries. OCT image analysis showed that the extent of total ablated material reached up to 1 mm3 as planned. The highest material removal rate with the laser was estimated at 1.65 x 10-3 mm3/s, which is lower than necessary for arthroscopic surgery. H&E and safranin-O staining displayed changes in tissue structure and proteoglycan loss only in tissue immediately surrounding sites of tissue removal. SHG imaging measured average susceptibility ratio and vector map which shows the projection of the in-out plane fibrils onto a flat image. In cartilage adjacent to ablation sites the susceptibility ratio increased compared to healthy controls indicative of change in fibrillar organization or saw a loss of signal suggestive of structural changes within individual collagen fibrils. However, change in susceptibility ratio or SHG signal loss corresponded to damage evident in histological sections and did not extend beyond into tissue. Notable loss of signal was also observed in samples with mechanically drilled holes. Confocal Z-stacks were used to produce 3-dimensional surface regions of dead and live cells surrounding the area of removed tissue. Between 10-50 measurements were taken from each surface region. Damage induced by different removal methods varied significantly (1-way ANOVA, F2, N = 366, p < 0.001, d.f. = 2, 167). Laser removal resulted in significantly less damage (48.4 u00b1 15.1 u03bcm, n = 3) compared to mechanically drilled holes (475 u00b1 148 u03bcm, n = 3) (Bonferroni test, p < 0.01) and tissues removed with a biopsy punch (130 u00b1 96.1u03bcm, n = 3) (Bonferroni test, p < 0.01). SEM images of ablated graft beds illustrated sharp edges and a smooth bottom with no signs of thermal effects such as melting and cracking.DISCUSSION: Unlike mechanical methods of cartilage removal, ultrafast laser ablation produces minimal levels of collateral tissue damage while allowing for the customization graft beds. Although the results of the laseru2013cartilage interaction with the ultra-fast burst laser are promising, the current laser systemu2019s ablation rate is to slow. However, planned modifications of the laser system should result in an increase of removal rate by a factor of 100. This will allow for much larger volumes of tissue to be removed in less time, meeting the requirements for arthroscopic procedures. Next steps are in-vitro evaluation of the ablation effects after culturing the cartilage explants for a week to capture later effects. Planned future experiments also include performing grafting procedures in live animals to compare the fit, remodeling, and healing of grafts prepared with the ultrafast laser to mechanical graft preparation.SIGNIFICANCE/CLINICAL RELEVANCE: Improvement in the precision of graft cutting and graft bed preparation could positively affect the surgical outcome of millions of patients with osteoarthritis. This work to date demonstrates the advantages of using an ultrafast fibre laser over mechanical tools, highlighting the potential clinical benefits of using such a technique for orthopedic surgery.ACKNOWLEDGEMENTS: Grant funding for this study was provided by the Canadian Institutes of Health Research (CIHR).Laser Drill Biopsy PunchREFERENCES:1. Ronn K, Reischl N, Gautier E, et al. 2011. Current surgical treatment of knee osteoarthritis. Arthritis 2011:454873.2. Akens MK, von Rechenberg B, Bittmann P, et al. 2001. Long term in-vivo studies of a photo-oxidized bovine osteochondral transplant in sheep. BMC Musculoskelet Disord 2.3. Imhoff AB. 1995. The Use of Lasers in Orthopaedic Surgery. Operative Techniques in Orthopaedics 5:192-203.Figure 1: Comparison of laser ablation to mechanical cutting of articular cartilage. A live/dead fluorescent assay labeled nucleic acid of damaged cells red while live cells transform and retain a green fluorophore.Bar = 200 u03bcmORS 2019 Annual Meeting Poster No. 0461

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