Porcine Septal Cartilage Research Articles

Zone-Dependent Architecture and Biochemical Composition of Decellularized Porcine Nasal Cartilage Modulate the Activity of Adipose Tissue-Derived Stem Cells in Cartilage Regeneration.

Previous anatomical studies have shown different functional zones in human nasal septal cartilage (NC). These zones differ in respect to histological architecture and biochemical composition. The aim of this study was to investigate the influence of these zones on the fate of stem cells from a regenerative perspective. Therefore, decellularized porcine septal cartilage was prepared and subjected to histological assessment to demonstrate its equivalence to human cartilage. Decellularized porcine NC (DPNC) exposed distinct surfaces depending on two different histological zones: the outer surface (OS), which is equivalent to the superficial zone, and the inner surface (IS), which is equivalent to the central zone. Human adipose tissue-derived stem cells (ASCs) were isolated from the abdominal fat tissue of five female patients and were seeded on the IS and OS of DPNC, respectively. Cell seeding efficiency (CSE), vitality, proliferation, migration, the production of sulfated glycosaminoglycans (sGAG) and chondrogenic differentiation capacity were evaluated by histological staining (DAPI, Phalloidin, Live-Dead), biochemical assays (alamarBlue®, PicoGreen®, DMMB) and the quantification of gene expression (qPCR). Results show that cell vitality and CSE were not influenced by DPNC zones. ASCs, however, showed a significantly higher proliferation and elevated expression of early chondrogenic differentiation, as well as fibrocartilage markers, on the OS. On the contrary, there was a significantly higher upregulation of hypertrophy marker MMP13 (p < 0.0001) and GAG production (p = 0.0105) on the IS, whereas cell invasion into the three-dimensional DPNC was higher in comparison to the OS. We conclude that the zonal-dependent distinct architecture and composition of NC modulates activities of ASCs seeded on DPNC. These findings might be used for engineering of cartilage substitutes needed in facial reconstructive surgery that yield an equivalent histological and functional structure, such as native NC.

Experimental approach to nasal septal cartilage regeneration with adipose tissue-derived stem cells and decellularized porcine septal cartilage.

Cartilage shortage is a major problem in facial reconstructive surgery. Prior studies have shown that decellularized porcine nasal septal cartilage (DPNC) seeded with primary human nasal chondrocytes enabled cartilage regeneration and showed potential as a replacement material for nasal cartilage. Since adipose tissue-derived stem cells (ASCs) are easily accessible and almost abundantly available, they appear to be a promising alternative to limited chondrocytes making the combination of DPNC and ASCs a feasible approach towards clinical translation. Thus, this study was intended to investigate the interactions between ASCs and DPNC in an in vitro model. DPNCs were seeded and 3D-cultured with primary human ASCs that were priorly characterized with trilineage differentiation and flow cytometry. Cell vitality and proliferation were evaluated by Live-Dead, alamarBlue, and PicoGreen assays. Chondrogenic differentiation was examined by DMMB assay and cryosectioning-based histology. Cell invasion within DPNC was visualized and quantified by fluorescent histology (DAPI, Phalloidin). ASCs showed good adherence to DPNC and Live-Dead assay proved their viability over 2weeks. AlamarBlueassay showed an increase in metabolic activity compared to 2D cultures, and PicoGreen assay demonstrated an increase of cell number within DPNC over time. Biochemical assays and histology added evidence of chondrogenic differentiation of 3D-cultured ASCs under the influence of chondrogenic induction medium. Fluorescent image analysis showed a significant increase of cell-occupied areas of scaffolds over time (P<.05). DPNC scaffolds provided a suitable environment for ASCs that allowed good cell vitality, high proliferation, and chondrogenic differentiation. Thus, the use of ASCs and DPNC yields a promising alternative to the use of primary human chondrocytes. For facial cartilage tissue engineering, we regard ASCs as an attractive alternative to human nasal chondrocytes due to their better accessibility and availability. Further research will be necessary to determine long-term effects and in vivo outcomes of ASCs and DPNC in cartilage regeneration of the face.

Temperature dependent change in equilibrium elastic modulus after thermally induced stress relaxation in porcine septal cartilage

Laser cartilage reshaping (LCR) is a promising method for the in situ treatment of structural deformities in the nasal septum, external ear and trachea. Laser heating leads to changes in cartilage mechanical properties and produces relaxation of internal stress allowing formation of a new stable shape. While some animal and preliminary human studies have demonstrated clinical feasibility of LCR, application of the method outside specialized centers requires a better understanding of the evolution of cartilage mechanical properties with temperature. The purpose of this study was to (1) develop a method for reliable evaluation of mechanical changes in the porcine septal cartilage undergoing stress relaxation during laser heating and (2) model the mechanical changes in cartilage at steady state following laser heating. Rectangular cartilage specimens harvested from porcine septum were heated uniformly by a radio-frequency (RF) electric field (500 kHz) for 8 and 12 seconds to maximum temperatures from 50 to 90 degrees C. Cylindrical samples were fashioned from the heated specimens and their equilibrium elastic modulus was measured in a step unconfined compression experiment. Functional dependencies of the elastic modulus and maximum temperature were interpolated from the measurements. Profiles of the elastic modulus produced after 8 and 12 seconds of laser irradiation (Nd:YAG, lambda = 1.34 microm, spot diameter 4.8 mm, laser power 8 W) were calculated from interpolation functions and surface temperature histories measured with a thermal camera. The calculated elastic modulus profiles were incorporated into a numerical model of uniaxial unconfined compression of laser irradiated cylindrical samples. The reaction force to a 0.1 compressive strain was calculated and compared with the reaction force obtained in analogous mechanical measurements experiment. RF heating of rectangular cartilage sample produces a spatially uniform temperature field (temperature variations < or = 4 degrees C) in a central region of the sample which is also large enough for reliable mechanical testing. Output power adjustment of the RF generator allows production of temperature histories that are very similar to those produced by laser heating at temperatures above 60 degrees C. This allows creation of RF cartilage samples with mechanical properties similar to laser irradiated cartilage, however with a spatially uniform temperature field. Cartilage equilibrium elastic modulus as a function of peak temperature were obtained from the mechanical testing of RF heated samples. In the temperature interval from 60 to 80 degrees C, the equilibrium modulus decreased from 0.08+/- 0.01 MPa to 0.016+/-0.007 MPa, respectively. The results of the numerical simulation of uniaxial compression of laser heated samples demonstrate good correlation with experimentally obtained reaction force. The thermal history and corresponding thermally induced modification of mechanical properties of laser irradiated septal cartilage can be mimicked by heating tissue samples with RF electric current with the added advantage of a uniform temperature profile. The spatial distribution of the mechanical properties obtained in septal cartilage after laser irradiation could be computed from mechanical testing of RF heated samples and used for numerical simulation of LCR procedure. Generalization of this methodology to incorporate orthogonal mechanical properties may aid in optimizing clinical laser cartilage reshaping procedures.

Stress Relaxation in Porcine Septal Cartilage During Electromechanical Reshaping: Mechanical and Electrical Responses

Electromechanical reshaping (EMR) of facial cartilage has recently been developed as an alternative to classic surgical techniques to alter cartilage shape. This study focuses on determining the underlying physical mechanisms responsible for shape change (stress relaxation) in mechanically deformed facial cartilage specimens exposed to constant electric fields. Flat porcine nasal septal cartilage specimens were deformed by an aluminum jig into semicylindrical shapes while a constant electric voltage was applied to the concave and convex surfaces of the specimen. Mechanical stress, electric current and resistance were measured during voltage application. Specimen shape retention was measured as retained bend angle. Total electric charge transferred in the electric circuit was calculated from the electric current measurement. Electrical resistance, transferred charge and the bend angle increase with increase in voltage application time until bend angle reaches maximum value determined by the jig geometry. Then, the bend angle decreases and electrical parameters nearly saturate. The time dependent behavior of electric current was analyzed using the Cottrell equation. The observed changes in electric current suggest that during the initial 1-2 min of EMR nonlinear diffusion determines electro-chemical reaction rates, which are then followed by a linear diffusion dominated process. Close correlation between alteration of cartilage mechanical state and change in its electrical properties suggest that an electro-chemical reaction is the dominant mechanism behind EMR.

Acoustic response of cartilage during laser-mediated stress relaxation

In this study, laser reshaping of porcine septal cartilage was performed using an Nd:YAG laser (/spl lambda/=1.32 /spl mu/m), while changes in acoustic waves were observed, in an attempt to develop technique to monitor the reshaping process. Concurrent measurements of strain (during tensile compression and tension, as well as flexure), temperature, and a 5-MHz ultrasonic signal were recorded during laser irradiation (/spl lambda/=1.32 /spl mu/m, 4 W, 13 s). The sample was set up in a water bath to enhance acoustic coupling. From the ultrasonic signal, both time of flight (TOF) and signal amplitude as reflected from the back wall of the cartilage were extracted and correlated with temperature and strain measurements. The onset of stress relaxation of the cartilage generally occurred between 50/spl deg/C and 60/spl deg/C. While TOF measurements indicated a generally constant increase in the speed of sound of the cartilage during the irradiation period, the amplitude of the reflected acoustic signal correlated directly with the stress relaxation of the cartilage. At the point of stress relaxation, the amplitude of the acoustic signal consistently attenuated to roughly 50% of its original magnitude.

Measurement of the elastic modulus of porcine septal cartilage specimens following Nd: YAG laser treatment.

Laser cartilage reshaping is a temperature-dependent process that results in stress relaxation with subsequent formation of a new and stable specimen geometry. The objective of this study was to quantitatively measure changes in the elastic moduli of porcine cartilage following laser heating. The elastic modulus of porcine nasal septal cartilage specimen (25 x 5 x 2 mm) was measured before and after Nd: YAG laser (lambda=1.32 Irvinem, 21.22 W/cm2) irradiation and following rehydration in saline solution. Specimens were secured in a single beam cantilever configuration and displaced using a calibrated thin beam load cell attached to a motorised micropositioner. Elastic modulus was calculated using elastic beam theory. Measurements were recorded before and immediately after laser heating, and following rehydration in saline solution (40 minutes, 25 degrees C). Specimens heated in saline (100 degrees C and then re-hydrated) were used as controls to determine the effect of total thermal denaturation. The calculated moduli before and after irradiation were 4.86 +/- .145 MPa and 1.166 +/- .055 MPa respectively. Following rehydration in saline, the modulus returned to near-baseline values (5.119 +/- .163 MPa). In contrast, elasticity remained lower in specimens boiled and re-hydrated (3.25 +/- .130 MPa). These findings suggest that cartilage matrix does not undergo complete thermal denaturation during laser reshaping, given the return in tissue properties with rehydration.

Radiofrequency cartilage reshaping: efficacy, biophysical measurements, and tissue viability.

To assess the feasibility of reshaping cartilage using radiofrequency (RF) heating, and to examine the effects of this process on tissue biophysical properties (optical and thermal) and cellular viability. Mechanically deformed porcine septal cartilage was reshaped using 2 RF-generating devices. We performed dynamic measurements of tissue thermal and optical properties while heating cartilage with one of these devices. Cellular viability was assessed immediately and 7 days after treatment. A characteristic change in the diffuse transmittance of light through the cartilage occurred during heating. Change in transmittance has been shown to accompany the onset of stress relaxation in cartilage. Peak radiometric surface temperature during heating was 88.6 degrees C. Specimens retained their user-specified curved shape for the observed period of 14 days. Chondrocyte viability in RF-heated tissue was 19% and 14% of that in untreated control specimens at days 0 and 7 after treatment, respectively. Radiofrequency heating has been shown to effectively reshape cartilage while maintaining cellular viability, illustrating a novel application for a widely used technology.

Shape retention in porcine-septal cartilage following Nd:YAG (lambda = 1.32 microm) laser-mediated reshaping.

Photothermal heating of mechanically deformed cartilage accelerates stress relaxation and results in sustained shape change. In this study, shape retention was measured in Nd:YAG laser reshaped porcine septal cartilage. Specimens were laser reshaped either 4 (Group I) or 28 hours (Group II) following extraction from the crania. Specimens were bent into approximately semicircular shapes and irradiated half way between the endpoints of the semicircle. Resultant bend angle was calculated based on linear measurements. Shape retention was calculated by comparing resultant curvature with pre-irradiation measurements. Mechanical deformation alone resulted in initial bend angles varying from 188 degrees to 229 degrees. Resultant bend angles varied from 84 degrees to 194 degrees corresponding to shape retention varying from 58 to 75%. Non-irradiated cartilage retained less than 46% of the original bend. Shape retention was greater in Group II, compared to Group I. In Group I, no cephalocranial difference in shape retention was observed, though in Group II greater shape retention was observed in rostral specimens. While laser heating does significantly reshape cartilage, clinical use of this technology will require "overbending" of the cartilage graft to compensate for this memory effect. The degree of overbending is likely to vary with cartilage type and location.

Laser‐mediated cartilage reshaping with feedback‐controlled cryogen spray cooling: Biophysical properties and viability

Background and Objective Recent studies have indicated that chondrocyte viability decreases with prolonged or repeated laser irradiation. To optimize laser-mediated cartilage reshaping, the heating process must be finely controlled. In this study, we use high-power Nd:YAG laser irradiation (λ = 1.32 μm) combined with cryogen spray cooling (CSC) in an attempt to reshape porcine septal cartilage while enhancing chondrocyte viability. Study Design/Materials and Methods Chondrocyte viability was determined after high-power (50 W/cm2) Nd:YAG-mediated cartilage reshaping with and without cryogen spray cooling (CSC) and correlated with dynamic measurements of tissue optical and thermal properties. Results After 1.5 to 2.0 seconds of laser exposure, characteristic changes in diffuse reflectance (indicating the onset of accelerated stress relaxation) was observed in both laser only and laser with CSC specimens. After 2 seconds of laser exposure, specimens in both groups retained the curved shape for up to 14 days. After one laser exposure, chondrocyte viability was 94.35 ± 6.1% with CSC and 68.77 ± 20.1% (P < 0.05) without CSC. After two laser exposures, a similar trend was observed with CSC (70.18 ± 16.44%) opposed to without CSC (28 ± 45%; P < 0.05). Conclusion CSC during high-power laser irradiation allows rapid heating while minimizing extreme front surface temperature elevations and axial thermal gradients. Laser irradiation with CSC can be used to effectively reshape cartilage tissue with the additional advantage of increasing chondrocyte viability. Lasers Surg. Med. 28:1–10, 2001. © 2001 Wiley-Liss, Inc.

Laser-mediated cartilage reshaping with feedback-controlled cryogen spray cooling: biophysical properties and viability.

Recent studies have indicated that chondrocyte viability decreases with prolonged or repeated laser irradiation. To optimize laser-mediated cartilage reshaping, the heating process must be finely controlled. In this study, we use high-power Nd:YAG laser irradiation (lambda = 1.32 microm) combined with cryogen spray cooling (CSC) in an attempt to reshape porcine septal cartilage while enhancing chondrocyte viability. Chondrocyte viability was determined after high-power (50 W/cm2) Nd:YAG-mediated cartilage reshaping with and without cryogen spray cooling (CSC) and correlated with dynamic measurements of tissue optical and thermal properties. After 1.5 to 2.0 seconds of laser exposure, characteristic changes in diffuse reflectance (indicating the onset of accelerated stress relaxation) was observed in both laser only and laser with CSC specimens. After 2 seconds of laser exposure, specimens in both groups retained the curved shape for up to 14 days. After one laser exposure, chondrocyte viability was 94.35 +/- 6.1% with CSC and 68.77 +/- 20.1% (P < 0.05) without CSC. After two laser exposures, a similar trend was observed with CSC (70.18 +/- 16.44%) opposed to without CSC (28 +/- 45%; P < 0.05). CSC during high-power laser irradiation allows rapid heating while minimizing extreme front surface temperature elevations and axial thermal gradients. Laser irradiation with CSC can be used to effectively reshape cartilage tissue with the additional advantage of increasing chondrocyte viability.

Stress Relaxation of Porcine Septal Cartilage During Nd:YAG (λ=1.32 μm) Laser Irradiation: Mechanical, Optical, and Thermal Responses

Laser-assisted cartilage reshaping is mediated by thermally induced stress relaxation, and may be used to alter cartilage morphology for reconstructive surgical procedures in the upper airway and face without carving, morselizing, or suturing. Internal stress σ(t), integrated backscattered light intensity I(t) from a He-Ne probe laser (λ=632.8 nm), and radiometric surface temperature Sc(t) were measured during the reshaping of porcine nasal septal cartilage using a pulsed Nd:YAG laser (λ=1.32 μm). Internal stress and integrated backscattered light intensity were observed to increase, plateau, and then decrease in similar ways during laser irradiation. The plateau region occurred when the cartilage front surface temperature approached 65 °C. I(t) was utilized in a feedback control procedure to reshape cartilage specimens from a flat to a curved geometry. Immediately following laser irradiation, the tissues were rehydrated in normal saline for 15 min while wrapped around a small dowel. A stable shape change was retained for 21 days while the specimens were stored in normal saline at 5 °C. The backscattered light intensity signal mirrors underlying changes in internal stress, and further rate of change or slope of I(t) is nearly zero when the surface temperature reaches about 65 °C. Measurements of I(t) (or, equivalently, the fractional change in integrated backscattered light intensity ΔI(t)/I0) may be used to control the process of laser-assisted cartilage reshaping and minimize nonspecific thermal injury due to uncontrolled heating. © 1998 Society of Photo-Optical Instrumentation Engineers.

Poster 28: Stereological Quantitation of Chondrocyte Volume Fraction Distribution in Porcine Septal Cartilage

Poster 28: Stereological Quantitation of Chondrocyte Volume Fraction Distribution in Porcine Septal Cartilage