Rabbit Nasal Septal Cartilage Research Articles

Comparison of Decellularized and Gamma-Irradiated Septal and Costal Cartilage Allografts in a Rabbit Model.

Background: Traditional gamma-irradiated allograft costal cartilage used for cartilage-depleted rhinoplasty patients contains cellular remnants that are potentially responsible for immunological stimulation and graft resorption. Objective: To determine whether decelullarized and/or chemically crosslinked rabbit costal and nasal septal cartilage allografts reduce the risk of allograft resorption. Materials and Methods: In vitro and in vivo analyses of septal and costal cartilage grafts in New Zealand white rabbits were carried out. Irradiated, decellularized, and/or carbodiimide crosslinked cartilage grafts were compared with nontreated autografts and allografts controls. Gross analysis, biomechanical testing, DNA quantification, and histological analyses were performed. Results: All treated grafts had a similar "feel" to native cartilage except for crosslinked grafts, which were significantly stiffer with decreased maximum load and tensile strain. Decellularization effectively reduced DNA content. Biomechanics of explants were unchanged except in untreated allografts, which exhibited increased stiffness, decreased strain, and significant scarring/fibrosis. There was increased glycosaminoglycans retention and less resorption in crosslinked septal cartilage grafts. Conclusion: This study demonstrates the potential benefits of decellularization and crosslinking allograft cartilage and further refinements in crosslinking may improve resorption characteristics while maintaining suitable physical characteristics.

Fibrin-Genipin Hydrogel for Cartilage Tissue Engineering in Nasal Reconstruction.

Nasal reconstruction is limited by the availability of autologous cartilage. The aim is to investigate an adhesive biomaterial for tissue engineering of nasal cartilage by evaluating mechanical properties of hydrogels made of fibrin crosslinked with genipin as compared to native tissue. Hydrogels of fibrin, fibrin-genipin, and fibrin-genipin with extracellular matrix (ECM) particles were created and evaluated with mechanical testing to determine compression, tensile, and shear properties. Rabbit nasal septal cartilage was harvested and tested in these modalities for comparison. Transmission electron microscopy characterized hydrogel structure. Fibrin-genipin gels had higher compressive, tensile, and shear moduli compared to fibrin alone or fibrin-genipin with ECM. However, all hydrogel formulations had lower moduli than the rabbit nasal septal cartilage. Electron microscopy showed genipin crosslinking increased structural density of the hydrogel and that cartilage ECM created larger structural features with lower crosslinking density. The addition of genipin significantly improved mechanical properties of fibrin hydrogels by increasing the compressive, tensile, and shear moduli. The addition of cartilage ECM, which can add native structure and composition, resulted in decreased moduli values. Fibrin-genipin is a bioactive and biomechanically stable hydrogel that may offer promise as a scaffold for cartilage tissue engineering in nasal reconstruction, yet further augmentation is required to match material properties of native nasal cartilage.

Anatomy and Surgical Approaches to the Rabbit Nasal Septum

The rabbit is the primary animal model used to investigate aspects of nasal surgery. Although several studies have used this model, none has provided a comprehensive analysis of the surgical anatomy and techniques used to gain access to the rabbit nasal fossae and septum. To describe and optimize the surgical anatomy and approach to the rabbit nasal vault and septal cartilage. In an ex vivo animal study conducted at an academic medical center, preliminary cadaveric dissections were performed on rabbit head specimens to establish familiarity with relevant anatomy and rehearse various approaches. Live Pasteurella-free New Zealand white rabbits (3.5-4.0 kg) were used to further develop this surgical technique developed here. Access of the nasal vault was gained through a midline nasal dorsum incision and creation of an osteoplastic flap with a drill. Submucosal resection was performed with preservation of the mucoperichondrium. All rabbits were monitored daily for 4 weeks in the postoperative period for signs of infection, pain, and complications. The study was conducted from June 1, 2014, to December 1, 2014. Surgical anatomy and techniques used to gain access to the rabbit nasal vault and harvest septal cartilage. Four Pasteurella-free New Zealand white rabbits (Western Organ Rabbit Co), ranging in age from 9 to 12 months and weighing between 3.5 and 4.0 kg, were used in this study. Initial dissections demonstrated the feasibility of harvesting septal cartilage while preserving the mucoperichondrial envelope. Access to the nasal vault through this 3-osteotomy approach allowed for maximal exposure to the nasal cavity bilaterally while maintaining the integrity of the mucoperichondrium following septal cartilage harvest. The maximum amount of bulk, en bloc, cartilage harvested was 1.0 × 2.5 cm. Following surgical dissection, all animals maintained adequate airway patency and support to midface structures. Furthermore, all specimens preserved the integrity of the mucoperichondrium, septum, vascular anatomy, and airway dynamics. No operative complications, postoperative airway compromise, or infections were observed. Access to the rabbit nasal vault and septal cartilage is feasible through a variety of surgical approaches and techniques. To date, this is the first study to meticulously document and review the surgical approaches to the rabbit nasal cavity. This approach describes a novel, 3-osteotomy method of accessing the nasal cavity bilaterally and successfully harvesting rabbit septal cartilage in a submucoperichondrial plane. The ability to preserve native anatomy and function allows for improved outcomes in translational and animal guided clinical research. NA.

In‐depth analysis of pH‐dependent mechanisms of electromechanical reshaping of rabbit nasal septal cartilage

Electromechanical reshaping (EMR) involves reshaping cartilage by mechanical deformation and delivering electric current to the area around the bend axis, causing local stress relaxation and permanent shape change. The mechanism of EMR is currently unclear, although preliminary studies suggest that voltage and application time are directly related to the concentration and diffusion of acid-base products within the treated tissue with little heat generation. This study aims to characterize local tissue pH changes following EMR and to demonstrate that local tissue pH changes are correlated with tissue damage and shape change. Ex vivo animal study involving EMR of rabbit nasal septal cartilage and biochemical estimation of tissue pH changes. The magnitude and diffusion of acid-base chemical products in control (0V, 2 minutes), shape change (4V, 4 minutes; 6V, 1, 2, 4 minutes; 8V, 1, 2 minutes), and tissue damage (8V, 4, 5 minutes; 10V, 4, 5 minutes) parameters following EMR are approximated by analyzing local pH changes after pH indicator application. There is a direct relationship between total charge transfer and extent of acid-base product diffusion (P <0.05). A "pH transition zone" is seen surrounding the bend apex above 8V, 2 minutes. Colorimetric analysis suggests that small local pH changes (10(-8) hydrogen ions) are at least partly implicated in clinically efficacious EMR. These results provide additional insight into the translational applications of EMR, particularly the relationship among pH changes, shape change, and tissue injury, and are integral in optimizing this promising technology for clinical use.

Effects of Different Levels of Crushing on the Viability of Rabbit Costal and Nasal Septal Cartilages

Cartilage grafts are frequently used in nasal surgery for structural and/or aesthetic purposes. The literature holds contradictory reports concerning the effect of crushing on the viability of cartilage grafts. Nasal septal and costal cartilage grafts were harvested from 12 New Zealand rabbits. Each nasal septal and costal cartilage was divided into five equal pieces. One of the pieces was left intact and the remaining four were prepared as slightly, moderately, severely, or significantly crushed. The cartilage pieces were then autoimplanted into the paravertebral skin of the rabbits. The animals were euthanized 4 months later and the effect of crushing on cartilage grafts was assessed pathologically. The viability of the chondrocytes was found to be decreased as the level of crushing increased. The mean chondrocyte viability rates for the intact, slightly crushed, moderately crushed, severely crushed, and significantly crushed cartilages were 88, 75, 51, 41, and 13 percent for the septal cartilages and 94, 83, 62, 32, and 26 percent for the costal cartilages, respectively. The differences between the mean viability rates of septal and costal cartilage groups were statistically not significant. The level of crushing determines the rate of viability for the crushed cartilage. Viability rates and the clinical properties of the slightly crushed cartilage grafts at long-term follow-up may be similar to those of the intact cartilage grafts. However, severe or significant crushing leads to a decrement in the viability of the chondrocytes and may cause unpredictable degrees of volume loss at long-term follow-up.

Using optical coherence tomography to monitor effects of electromechanical reshaping in septal cartilage

Electromechanical reshaping (EMR) of cartilage is a promising noninvasive technique with potential for broad application in reconstructive surgery. EMR involves applying direct current electrical fields to localized stress regions and then initiating a series of oxidation-reduction reactions, thus effecting a shape change. Previous EMR studies have focused on macroscopic structural measurements of the shape change effect or monitoring of electrical current flow. Only limited investigation of structural changes in the tissue at the histologic level have been performed, and not in real time. This study is the first to use optical coherence tomography (OCT) to examine structural changes in cartilage during EMR. Two platinum needle electrodes were inserted into fixed rectangular rabbit nasal septal cartilage specimens. The spectral domain OCT probe was then positioned above the anode needle. A constant voltage of 6V was applied for 3 minutes, and images were obtained (8 frames/second). OCT was also performed in specimens undergoing dehydration under ambient conditions and during pH changes produced by the addition of HCl, as both processes accompany EMR. The OCT data identified distinct findings among the three conditions, suggesting that EMR causes a much greater degree of reshaping on a molecular level than dehydration or a change in pH alone. OCT provides a means to gauge structural changes in the tissue matrix during EMR. The application of OCT to image the EMR process will add to our understanding of the mechanisms of action involved and potentially facilitate optimization of this process.

Survival of Chondrocytes in Rabbit Septal Cartilage After Electromechanical Reshaping

Electromechanical reshaping (EMR) has been recently described as an alternative method for reshaping facial cartilage without the need for incisions or sutures. This study focuses on determining the short- and long-term viability of chondrocytes following EMR in cartilage grafts maintained in tissue culture. Flat rabbit nasal septal cartilage specimens were bent into semi-cylindrical shapes by an aluminum jig while a constant electric voltage was applied across the concave and convex surfaces. After EMR, specimens were maintained in culture media for 64 days. Over this time period, specimens were serially biopsied and then stained with a fluorescent live–dead assay system and imaged using laser scanning confocal microscopy. In addition, the fraction of viable chondrocytes was measured, correlated with voltage, voltage application time, electric field configuration, and examined serially. The fraction of viable chondrocytes decreased with voltage and application time. High local electric field intensity and proximity to the positive electrode also focally reduced chondrocyte viability. The density of viable chondrocytes decreased over time and reached a steady state after 2–4 weeks. Viable cells were concentrated within the central region of the specimen. Approximately 20% of original chondrocytes remained viable after reshaping with optimal voltage and application time parameters and compared favorably with conventional surgical shape change techniques such as morselization.

Needle Electrode-Based Electromechanical Reshaping of Cartilage

Electromechanical reshaping (EMR) of cartilage provides an alternative to the classic surgical techniques of modifying the shape of facial cartilages. The original embodiment of EMR required surface electrodes to be in direct contact with the entire cartilage region being reshaped. This study evaluates the feasibility of using needle electrode systems for EMR of facial cartilage and evaluates the relationships between electrode configuration, voltage, and application time in effecting shape change. Flat rabbit nasal septal cartilage specimens were deformed by a jig into a 90° bend, while a constant electric voltage was applied to needle electrodes that were inserted into the cartilage. The electrode configuration, voltage (0–7.5 V), and application time (1–9 min) were varied systematically to create the most effective shape change. Electric current and temperature were measured during voltage application, and the resulting specimen shape was assessed in terms of retained bend angle. In order to demonstrate the clinical feasibility of EMR, the most effective and practical settings from the septal cartilage experimentation were used to reshape intact rabbit and pig ears ex vivo. Cell viability of the cartilage after EMR was determined using confocal microscopy in conjunction with a live/dead assay. Overall, cartilage reshaping increased with increased voltage and increased application time. For all electrode configurations and application times tested, heat generation was negligible (<1 °C) up to 6 V. At 6 V, with the most effective electrode configuration, the bend angle began to significantly increase after 2 min of application time and began to plateau above 5 min. As a function of voltage at 2 min of application time, significant reshaping occurred at and above 5 V, with no significant increase in the bend angle between 6 and 7.5 V. In conclusion, electromechanical reshaping of cartilage grafts and intact ears can be effectively performed with negligible temperature elevation and spatially limited cell injury using needle electrodes.

Analysis of Nd:YAG laser‐mediated thermal damage in rabbit nasal septal cartilage

Laser cartilage reshaping (LCR) involves the use of photo-thermal heating to reshape cartilage. Its clinical relevance depends on the ability to minimize thermal injury in irradiated regions. The present study seeks to understand the safety of LCR by determining shape change and resultant tissue viability as a function of laser dosimetry. Rabbit nasal septal cartilage were irradiated using a Nd:YAG laser (lambda = 1.32 microm, 5.4 mm spot diameter) with different exposure times of 4, 6, 8, 10, 12, and 16 seconds and powers of 4, 6, and 8 W. Temperature on the cartilage surface in the laser-irradiated region was collected using infrared thermography, this data was then used to predict tissue damage via a rate process model. A Live/Dead viability assay combined with fluorescent confocal microscopy was used to measure the amount of thermal damage generated in the irradiated specimens. Considerable thermal injury occurred at and below the laser-reshaping parameters that produced clinically relevant shape change using the present Nd:YAG laser. Confocal microscopy identified dead cells spanning the entire cross-sectional thickness of the cartilage specimen (about 500 microm thick) at laser power density and exposure times above 4 W and 6 seconds; damage increased with time and irradiance. The damage predictions made by the rate process model compared favorably with measured data. These results demonstrate that significant thermal damage is concurrent with clinically relevant shape change. This contradicts previous notions that there is a privileged laser dosimetry parameter where clinically relevant shape change and tissue viability coexist.

Long‐term in vivo stability of rabbit nasal septal cartilage following laser cartilage reshaping: A pilot investigation

To evaluate the long-term effect of laser cartilage reshaping on rabbit nasal septal cartilage viability and mechanical integrity in an in vivo model. In vivo animal investigation. Rabbit septal cartilage specimens were laser (Nd:YAG, lambda = 1.32 mum, spot size 5.4-mm diameter, 10 W, 10 seconds, 50 Hz PPR) reshaped and subsequently reimplanted into an interscapular subcutaneous pocket. Specimens were harvested at 8 and 12 months and evaluated using photography, flow cytometry, and histology. Grossly, specimens showed alteration in the physical integrity with varying degrees of tissue resorption. The non-irradiated control specimens demonstrated significantly increased stiffness. Histologically, there was marked depletion of the extracellular matrix and an overall reduction in tissue mass in laser irradiated tissues. However, flow cytometry data identified viable chondrocytes in laser-irradiated specimens that were identical to those observed in controls. Study results demonstrate that the rabbit nasal septal cartilage model can be effectively used to study laser reshaping, however alternative recipient sites with perichondrial lining, such as the pinna, may provide a more realistic physiologic environment for reshaped graft tissue. The dosimetry used in this pilot study likely led to significant thermal injury. Study results underscore the importance of elucidating the optimal laser dosimetry required to initiate permanent shape change while minimizing thermal damage.

Shape retention in porcine and rabbit nasal septal cartilage using saline bath immersion and Nd:YAG laser irradiation

The process of altering the shape of cartilage using heat has been referred to as thermoforming, and presents certain clinical benefits in reconstructive surgical procedures within the head and neck. Thermoforming allows cartilage in the upper airway and face to be reshaped without the use of classic surgical maneuvers such as carving, morselizing, or suturing. The goal of this study was to determine the dependence of cartilage shape change on both temperature and laser dosimetry using two thermoforming methods: saline bath immersion and laser irradiation. Ex-vivo rabbit and porcine nasal septal cartilages were mechanically deformed and reshaped using the two thermoforming methods. With saline bath immersion using rabbit cartilage, each specimen was deformed by securing it to a small copper tube (outer diameter 8 mm) using dental bands. For porcine cartilage immersed in a saline bath, each sample was mechanically deformed between two pieces of wire mesh attached to a semicircular acrylic block. With both porcine and rabbit cartilage, the specimen and apparatus were then immersed in a hot saline bath for time intervals varying from 20 and 320 seconds and at constant temperatures between 62 and 74 degrees C. In laser reshaping, the cartilage specimens were mechanically deformed on a jig and consecutively irradiated with an Nd:YAG laser (lambda = 1.32 microm) in several spots for 6-16 seconds and irradiances of 10.2-40.7 W/cm2 per spot. After either saline bath heating or irradiation, cartilage specimens were immersed in room temperature saline for 15 minutes, then upon removal from the jig the length between the ends of each specimen was measured in order to calculate the resulting bend angle. The transition zone for cartilage reshaping was defined as where a significant increase in bend angle was observed between consecutive times of immersion/irradiation at the same temperature/irradiance. For the saline bath experiments, the transition zone was observed between 59-68 degrees C and 62-68 degrees C for porcine and rabbit cartilage, respectively. Similar transition zones occurred with laser irradiation below irradiances of 20.4 W/cm2 for both porcine and rabbit cartilage. In addition, the dosimetry pairs in the transition zones produce peak temperatures below the thresholds determined from the saline bath immersion studies. The critical transition temperature region was determined by the sharp increase in bend angle at consecutive times of immersion at the same temperature. This range was determined to be 59-68 degrees C and 62-68 degrees C for porcine and rabbit cartilage, respectively. Similar transition zones for dosimetry occurred below 20.4 W/cm2 during cartilage irradiation in both species.

Identification of chondrocyte proliferation following laser irradiation, thermal injury, and mechanical trauma

Cartilage has a limited regenerative capacity, and there are a lack of reliable techniques and methods to stimulate growth of new tissue to treat degenerative diseases and trauma. This study focused on identifying chondrocyte cell proliferation in ex vivo cartilage tissue following heating Nd:YAG laser using whole-mount analysis and flow cytometry, and compared findings with results produced by contact, and water bath heating methods, mechanical injury, and the addition of transforming growth factor-beta (TGF-beta). Ex vivo rabbit nasal septal cartilages were either irradiated with an Nd:YAG laser (lambda = 1.32 microm, 2-16 seconds, 6 W/cm(2)), heated by immersion in a warm saline bath, heated by direct contact with a metal rod, or mechanically damaged by scoring with a scalpel or crushing. After treatment, specimens were incubated for 7 or 14 days in growth media containing 10 microM bromodeoxyuridine (BrdU). Additional specimens were cultured with both BrdU and TGF-beta. Both whole-mount BrdU-double-antibody detection techniques and flow cytometry were used to determine the presence of DNA replication as a marker of proliferation. An annular region of regenerating chondrocytes was identified surrounding the laser irradiation zone in whole-mount tissue specimens, and the diameter of this region increased with irradiation time. Using whole-mount analysis, no evidence of chondrocyte DNA replication was observed in tissues heated using non-laser methods, grown in TGF-beta, or mechanically traumatized. In contrast, flow cytometry identified the presence of BrdU-positive cells in the S-phase of the cell cycle (synthesis of DNA) for all protocols, indicating chondrocyte proliferation. The percentage of cells that are in S-phase increased with irradiation time. These data provide evidence that laser irradiation, along with other thermal and mechanical treatments, causes a proliferative response in chondrocytes, and this is observed ex vivo in the absence of cellular and humoral repair mechanisms. The advantage of using optical methods to generate heat in cartilage is that microspot injuries could be created in tissue and scanned across surfaces in clinical applications.

The viability of rabbit nasal septal cartilage following electromechanical reshaping

Problem: Electromechanical reshaping (electroforming) is a method used to alter the shape of native cartilage tissue by combining mechanical deformation and low current DC electric fields. The objective of this study was to evaluate the viability and spatial distribution of cellular injury in electromechanically reshaped rabbit nasal septal cartilage using confocal laser scanning microscopy (CLSM). Methods: Rabbit nasal septal cartilage grafts were harvested and placed between 2 semicylindrical aluminum electrodes. A DC electric field was established using various combinations of voltage (0–9 V) and application time (0–5 minutes). Intact grafts were stained with fluorescent indicators (SYTO-10 green stain for living cells, ethidium bromide red stain for dead cells), permitting the identification of cell morphology and viability within various regions. The overall degree of chondrocyte viability was correlated with voltage, application time, current density, and electrode polarity. Results: Increasing voltage and/or application time resulted in a greater degree of chondrocyte death, predominantly in the central region (highest current density) of specimens. More dead cells were observed near the negative electrode. Regions with no direct electrode contact consisted of mostly living cells. Conclusion: Cell viability in electroformed cartilage can be optimized with prudent selection of electrode geometry, voltage, and application time. Significance: CLSM provides a means of studying chondrocyte viability and insight into how electromechanical cartilage reshaping can be optimized. Support: National Institute of Health, the Air Force Office of Scientific Research, and the American Society of Lasers in Surgery and Medicine.

Measurement of the elastic modulus of rabbit nasal septal cartilage during Nd:YAG (lambda = 1.32 microm) laser irradiation.

The objective of this study was to quantitatively measure changes in the elastic moduli of rabbit nasal septal cartilage during laser heating. While the efficacy of laser cartilage reshaping has been established for use in nasal surgery, few studies have investigated the temperature-dependent viscoelastic behavior of cartilage. Cyclic force versus displacement curves were generated during the Nd:YAG laser (lambda = 1.32 microm, 10 second exposure time, 21.22 W/cm2) irradiation of cartilage specimens secured in cantilevered geometry. Samples were irradiated three times with 30 second cooling intervals between each laser exposure. Measurements were recorded before, during, and after laser irradiation, and then following complete rehydration in normal saline (NS) for 1 hour at 25 degrees C. Elastic modulus was calculated assuming linear viscoelastic behavior. The elastic modulus in native tissue decreased during and after successive laser exposures from about 6 to 3.5 MPa. After rehydration, the modulus returned to near-baseline value. Surface temperature reached a maximum of 65 degrees C. The laser irradiation of cartilage using parameters similar to those used in reshaping does not produce significant irreversible changes in the mechanical properties of the tissue. Measurement of the elastic modulus is an effective means of characterizing alterations in cartilage mechanical behavior during and after laser heating.

The porcine and lagomorph septal cartilages: models for tissue engineering and morphologic cartilage research.

Interest in reconstruction and modification of the facial cartilaginous frameworks using advanced technology and instrumentation is growing rapidly. Despite this maturing interest, no animal model has been established to provide morphologic cartilage tissue with similar characteristics to human septum in suitable quantities. The objective of this study was to characterize porcine and lagomorph (rabbit) nasal septal cartilage tissue. Both models share great similarity with their human counterpart and provide a low-cost, high-volume, and easily obtained source of bulk cartilage tissue. We present a technique for harvesting intact septal cartilages from these species, and characterize select cellular, metabolic, and physical properties using pulse-chase radiolabeling, flow cytometry, and mechanical analysis. Our selective evaluation of key tissue properties establishes these species as appropriate animal models for nasal septal cartilaginous surgery.

Periodate oxidation of acid polysaccharides

1. A two-step PAS method for acid mucopolysaccharides containing 1∶4 linked uronio acid (e.g. chondroitin sulphate) is described. A primary oxidation with sodium periodate oxidises rapidly reacting substrates. The aldehydes are reduced with aqueous sodium borohydride to Schiff-negative materials. A secondary, longer, oxidation step produces aldehyde from the slowly reacting acid mucopolysaccharides, which are demonstrated with Schiff. 2. The distribution of stain in the two-step PAS method in rabbit liver, human lung and nasal septal cartilage is as predicted from model experiments. 3. This, the first chemical method for localising acid mucopolysaccharides, is compared with “critical electrolyte concentration” techniques using Alcian blue for the same purposes. Preliminary data presented here show that both sets of results are compatible.