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Objectives Structural properties of tissue-engineered cartilage can be optimized by altering its collagen to sulfated glycosaminoglycan (sGAG) ratio with hyaluronidase. The objective was to determine if treatment of neocartilage constructs with hyaluronidase leads to increased collagen:sGAG ratios, as seen in native tissue, and improved tensile properties. Study Design Prospective, basic science. Methods Engineered human septal cartilage from 12 patients was treated with hyaluronidase prior to culture. Control and treated constructs were analyzed at 3, 6, or 9 weeks for their biochemical, biomechanical, and histological properties. Results Levels of sGAG were significantly reduced in treated constructs when compared with control constructs at 3, 6, and 9 weeks. Treated constructs had higher collagen:sGAG ratios when compared with control constructs at 3, 6, and 9 weeks. Treated constructs had greater tensile strength, strain at failure, and increased stiffness as measured by the equilibrium and ramp tensile moduli when compared with the untreated control constructs. Continued time in culture improved tensile strength in both treated and control constructs. Conclusion Hyaluronidase treatment of engineered septal cartilage decreased total sGAG content without inhibiting expansive growth of the constructs. Decreased sGAG in treated constructs resulted in increased collagen to sGAG ratios and was associated with an increase in tensile strength and stiffness. With additional culture time, sGAG increased modestly in depleted constructs, and some initial gains in tensile properties were dampened. Alterations in the dosage of hyalurondiase during neocartilage fabrication can create constructs that have improved biomechanical properties for eventual surgical implantation. Level of Evidence NA. Laryngoscope, 2016

Tissue engineering of human septal neocartilage offers the potential to produce large quantities of autologous material for use in the repair of cartilaginous craniofacial defects. Culture of septal neocartilage constructs in a rotary cell culture system bioreactor may improve the biochemical and biomechanical properties of engineered tissue. The objective of this study was to characterize the morphological, compositional and mechanical properties of engineered human septal cartilage constructs when cultured in three different environments. Human septal cartilage constructs were cultured in transwell plates for 6 weeks and subsequently cultured for an additional 4 weeks in transwell plates, free-floating in a sterile media bottle, or in a rotary cell culture system bioreactor. The histologic, biochemical, and biomechanical properties of the constructs were examined. All constructs maintained their radial dimensions throughout the culture period. Qualitatively, the constructs cultured in the rotary cell culture system bioreactor were firmer and more resilient compared with the constructs cultured under free-floating static and transwell plate conditions. Culture of human septal neocartilage constructs in a rotary cell culture system bioreactor augmented cell proliferation and extracellular matrix production when compared with constructs cultured in transwell plates and free-floating static conditions. Additionally, rotary cell culture system bioreactor culture enhanced total and type II collagen production. The rotary cell culture system bioreactor constructs possessed improved biomechanical properties compared with the other conditions and this is reflected in their superior resistance to manipulation. These findings suggest that culture in a rotary cell culture bioreactor can modulate the composition and mechanical function of engineered scaffold-free human septal cartilage constructs to produce constructs better suited for reconstructive surgery.

Objectives 1) To localize quantitatively the major biochemical constituents of native adult human septal cartilage across whole septa.

Tissue-engineered septal cartilage may provide a source of autologous cartilage for repair of nasal defects. Production of clinically useful neocartilage involves multiple steps that include manipulating the culture environment. Partial pressure of oxygen (ppO(2) ) is a property that has been shown to influence cartilage development. Specifically, studies suggest low ppO(2) augments in vitro growth of articular cartilage. Although in vivo measurements of articular cartilage ppO(2) have demonstrated hypoxic conditions, measurements have not been performed in septal cartilage. The objective of this study was to determine the ppO(2) of septal cartilage in vivo.Prospective, basic science.The ppO(2) was measured in 14 patients (mean ± standard deviation age, 35.9 ± 14.5 years; range, 18-63 years) during routine septoplasty or septorhinoplasty using the OxyLab pO(2) monitor (Oxford Optronix Ltd., Oxford, UK). Measurements were taken from the septum and inferior turbinate. Each patient's age and sex were recorded.The average ppO(2) measured at the septum and inferior turbinate was 10.5 ± 10.1 mm Hg (1.4 ± 1.3%) and 27.6 ± 12.4 mm Hg (3.6 ± 1.6%), respectively. The ppO(2) of these locations was significantly different (P.005). Advancing age was positively correlated with septal ppO(2) (R(2) = 0.42; P.05). Septal ppO(2) showed no significant sex variation.This is the first report of in vivo measurement of ppO(2) in septal cartilage. The data demonstrate reduced oxygenation of septal cartilage relative to the inferior turbinate. This elucidates an important characteristic of the in vivo milieu that can be applied to septal cartilage tissue engineering.

Structural properties of tissue-engineered cartilage can be optimized by altering its collagen to sulfated glycosaminoglycan (sGAG) ratio with hyaluronidase. The objective was to determine if treatment of neocartilage constructs with hyaluronidase leads to increased collagen:sGAG ratios, as seen in native tissue, and improved tensile properties.Prospective, basic science.Engineered human septal cartilage from 12 patients was treated with hyaluronidase prior to culture. Control and treated constructs were analyzed at 3, 6, or 9 weeks for their biochemical, biomechanical, and histological properties.Levels of sGAG were significantly reduced in treated constructs when compared with control constructs at 3, 6, and 9 weeks. Treated constructs had higher collagen:sGAG ratios when compared with control constructs at 3, 6, and 9 weeks. Treated constructs had greater tensile strength, strain at failure, and increased stiffness as measured by the equilibrium and ramp tensile moduli when compared with the untreated control constructs. Continued time in culture improved tensile strength in both treated and control constructs.Hyaluronidase treatment of engineered septal cartilage decreased total sGAG content without inhibiting expansive growth of the constructs. Decreased sGAG in treated constructs resulted in increased collagen to sGAG ratios and was associated with an increase in tensile strength and stiffness. With additional culture time, sGAG increased modestly in depleted constructs, and some initial gains in tensile properties were dampened. Alterations in the dosage of hyalurondiase during neocartilage fabrication can create constructs that have improved biomechanical properties for eventual surgical implantation.NA. Laryngoscope, 126:1984-1989, 2016.

Cartilage tissue engineering has the opportunity to profoundly impact clinical health care solutions for craniofacial reconstruction. Current reconstructive options are limited by inadequate autologous tissue availability, donor site morbidity, the potential for immune rejection, and even extrusion of allogenic or alloplastic grafts. Tissue-engineered cartilage would provide surgeons with an invaluable alternative tissue source. Current tissue engineering strategies begin with harvesting a small biopsy specimen from a patient. The chondrocytes are then isolated, expanded in monolayer culture, transferred to three-dimensional culture, and then matured in a dynamic culture apparatus called a bioreactor. Ultimately, the ideal tissue-engineered cartilage would be generated in shapes and sizes suited for craniofacial reconstruction, and would resemble native tissue in terms of its biochemical and structural properties. In this chapter, the process and state of the art of cartilage tissue engineering are described, as are the future challenges and opportunities within the field.

Tissue-engineered nasal septal cartilage may provide a source of autologous tissue for repair of craniofacial defects. Although advances have been made in manipulating the chondrocyte culture environment for production of neocartilage, consensus on the best oxygen tension for in vitro growth of tissue-engineered cartilage has not been reached. The objective of this study was to determine whether in vitro oxygen tension influences chondrocyte expansion and redifferentiation. Proliferation of chondrocytes from 12 patients expanded in monolayer under hypoxic (5% or 10%) or normoxic (21%) oxygen tension was compared over 14 days of culture. The highest performing oxygen level was used for further expansion of the monolayer cultures. At confluency, chondrocytes were redifferentiated by encapsulation in alginate beads and cultured for 14 days under hypoxic (5 or 10%) or normoxic (21%) oxygen tension. Biochemical and histological properties were evaluated. Chondrocyte proliferation in monolayer and redifferentiation in alginate beads were supported by all oxygen tensions tested. Chondrocytes in monolayer culture had increased proliferation at normoxic oxygen tension (p = 0.06), as well as greater accumulation of glycosaminoglycan (GAG) during chondrocyte redifferentiation (p < 0.05). Chondrocytes released from beads cultured under all three oxygen levels showed robust accumulation of GAG and type II collagen with a lower degree of type I collagen immunoreactivity. Finally, formation of chondrocyte clusters was associated with decreasing oxygen tension (p < 0.05). Expansion of human septal chondrocytes in monolayer culture was greatest at normoxic oxygen tension. Both normoxic and hypoxic culture of human septal chondrocytes embedded in alginate beads supported robust extracellular matrix deposition. However, GAG accumulation was significantly enhanced under normoxic culture conditions. Chondrocyte cluster formation was associated with hypoxic oxygen tension.

Objectives. (1) Evaluate safety of autogenous engineered septal neocartilage grafts and (2) compare properties of implanted grafts versus controls. Study Design. Prospective, basic science. Setting. Research laboratory. Methods. Constructs were fabricated from septal cartilage and then cultured in vitro or implanted on the nasal dorsum as autogenous grafts for 30 or 60 days. Rabbits were monitored for local and systemic complications. Histological, biochemical, and biomechanical properties of constructs were evaluated. Results. No serious complications were observed. Implanted constructs contained more DNA ( ) and less sGAG perDNA ( ) when compared with in vitro controls. Confined compressive aggregate moduli were also higher in implanted constructs ( ) and increased with longer in vivo incubation time ( ). Implanted constructs displayed resorption rates of 20–45 percent. Calcium deposition in implanted constructs was observe. Conclusion. Autogenous engineered septal cartilage grafts were well tolerated. As seen in experiments with athymic mice, implanted constructs accumulated more DNA and less sGAG when compared with in vitro controls. Confined compressive aggregate moduli were higher in implanted constructs. Implanted constructs displayed resorption rates similar to previously published studies using autogenous implants of native cartilage.

Volume Expansion of Tissue Engineered Human Nasal Septal Cartilage Importance: Cartilaginous craniofacial defects range in size and autologous cartilaginous tissue is preferred for repair of these defects. Therefore, it is important to have the ability to produce large size cartilaginous constructs for repair of cartilaginous abnormalities. Objectives: 1. To produce autologous human septal neocartilage constructs substantially larger in size than previously produced constructs 2. To demonstrate that volume expanded neocartilage constructs possess comparable histological and biochemical properties to standard size constructs 3. To show that volume expanded neocartilage constructs retain similar biomechanical properties to standard size constructs. Design: Prospective, basic science. Setting: Laboratory Participants: The study used remnant human septal specimens removed during routine surgery at the University of California, San Diego Medical Center or San Diego Veterans Affairs Medical Center. Cartilage from a total of 8 donors was collected.

Objectives:1) Produce autologous human septal neocartilage constructs substantially larger in size than previously produced constructs. 2) Demonstrate that volume expanded neocartilage constructs possess comparable histologic and biochemical properties to standard size constructs. 3) Show that volume expanded neocartilage constructs retain similar biomechanical properties to standard size constructs.Methods:Human septal chondrocytes from 8 donors were used to create 12mm and 24mm neocartilage constructs. These were cultured for a total of 10 weeks. Photo documentation, histologic, biochemical, and biomechanical properties were measured and compared.Results:The 24mm diameter constructs were qualitatively similar to the 12mm constructs. They possessed adequate strength and durability to be manually manipulated. Histologic analysis of the constructs demonstrated similar staining patterns in standard and volume expanded constructs. Proliferation, as measured by DNA content, was similar in 24mm and 12mm constr...

To test engineered and native septal cartilage for resistance to deformation and remodeling under sustained bending loads and to determine the effect of bending loads on the biochemical properties of constructs.Prospective, basic science.Laboratory.Human septal chondrocytes from 6 donors were used to create 12-mm constructs. These were cultured for 10 weeks and subjected to bending for 6 days. Free-swelling controls and native tissue from 6 donors were used for comparison. Shape retention, photo documentation, live-dead staining, and biochemical properties were measured.Live-dead staining showed no difference in cell survival between loaded constructs and free-swelling controls. The immediate shape retention of the constructs was 39.0% versus 24.4% for native tissue (P = .13). After 2 and 24 hours of relaxation, the constructs possessed similar shape retention to native tissue (26.9% and 16.4%; P = .126; 21.7% and 14.4%; P = .153). There was no significant change in construct shape retention from immediately after release to 2 hours of relaxation (39.0% and 26.9%, respectively; P = .238). In addition, the retention did not change significantly between 2 and 24 hours of relaxation (26.9% and 21.7%; P = .48). There was no significant difference in biochemical properties between loaded constructs and controls.The shape retention properties of human septal neocartilage constructs are comparable to human native septal cartilage. In addition, mechanical loading of neocartilage constructs does not adversely affect cell viability or biochemical properties. This study demonstrates that neocartilage constructs possess adequate shape fidelity for use as septal cartilage graft material.

ObjectiveTo determine and compare the bending moduli of native and engineered human septal cartilage.Study DesignProspective, basic science.SettingResearch laboratory.Subjects and MethodsNeocartilage constructs were fabricated from expanded human septal chondrocytes cultured in differentiation medium for 10 weeks. Constructs (n = 10) and native septal cartilage (n = 5) were tested in a 3-point bending apparatus, and the bending moduli were calculated using Euler-Bernoulli beam theory.ResultsAll samples were tested successfully and returned to their initial shape after unloading. The bending modulus of engineered constructs (0.32 ± 0.25 MPa, mean ± SD) was 16% of that of native septal cartilage (1.97 ± 1.25 MPa).ConclusionHuman septal constructs, fabricated from cultured human septal chondrocytes, are more compliant in bending than native human septal tissue. The bending modulus of engineered septal cartilage can be measured, and this modulus provides a useful measure of construct rigidity while undergoing...

The mAb lan3-6 recognizes a cytosolic antigen which is selectively expressed in the growth cones and axons of a small subset of peripheral sensory neurons fasciculating in a single tract common to all hirudinid leeches. We have used this antibody to clone a novel EF-hand calcium-binding protein, calsensin, by screening an expression vector library. A full-length clone of 1.1 kb identified by the antibody was isolated and sequenced. In situ hybridizations with calsensin probes and antibody staining using new polyclonal antisera generated against calsensin sequence demonstrate that calsensin indeed corresponds to the lan3-6 antigen. Calsensin consists of 83 residues with a calculated molecular mass of 9.1 kD that contains two helix-loop-helix domains. The calcium-binding domains are likely to be functional in vivo since a fusion protein derived from the calsensin clone binds 45Ca2+ in vitro. Immunoaffinity purification experiments with the lan3-6 antibody shows that a large 200,000 M(r) protein selectively copurifies with calsensin in two different leech species. These results suggest that calsensin may be functioning as a trigger protein which interacts with the larger protein. These data are consistent with the hypothesis that calsensin may mediate calcium-dependent signal transduction events in the growth cones and axons of this small group of sensory neurons which fasciculate in a single axon tract.

Objective: 1) To show that human septal neocartilage constructs possess comparable shape fidelity to native human septal tissue. 2) To demonstrate that the biochemical properties of human septal neocartilage constructs are unaffected by imposed bending and are equivalent to those of free-swelling controls.Method: Human septal chondrocytes from 9 donors were used to create 12-mm constructs. These were cultured for a total of 10 weeks and subjected to bending for 6 days. Free-swelling controls and native tissue were used for comparison. Shape retention, photo documentation, live-dead staining, and biochemical properties were measured.Results: Live-dead staining showed no difference in cell survival between shaped constructs and free-swelling controls. The immediate shape retention of the constructs was 39.0% versus 23.5% for native tissue (P = .06). After 2 hours of relaxation, the constructs possessed similar shape retention to native tissue (26.9% and 20.6%, respectively; P = .073). There was a significan...

To localize quantitatively the major biochemical constituents of native adult human septal cartilage across whole septa.Prospective, basic science.The nasal septa from seven cadavers were partitioned into 24 separate regions: six from caudal to cephalic and four from dorsal to ventral. Biochemical assays were used to determine the quantities, relative to wet weight, of the major constituents of cartilage: chondrocytes, collagen, and sulfated glycosaminoglycan.On average, each milligram of wet cartilage contained 24,900 cells, 73.9 μg collagen, and 17.1 μg sulfated glycosaminoglycan. Cell number showed no significant variation across the septa. In contrast, the caudal regions of the septa were associated with higher levels of collagen, the ventral regions correlated with higher levels of sulfated glycosaminoglycan, and the dorsal regions were associated with an elevated ratio of collagen to sulfated glycosaminoglycan.This study represents the first characterization of the biochemical composition of native human septal cartilage across whole septa. Quantities of collagen and sulfated glycosaminoglycan showed region-specific variation across the septum. The localized pattern of collagen and sulfated glycosaminoglycan deposition are consistent with the significance of preserving the L-strut during rhinoplasty and other nasal reconstructive procedures. In addition, it may assist in defining design goals for tissue-engineered septal neocartilage constructs to meet specific reconstructive needs in the future.

(1) To show that extracellular matrix deposition in 3-dimensional culture of human septal chondrocytes cultured in a rotary bioreactor is comparable to the deposition achieved under static culture conditions. (2) To demonstrate that the biomechanical properties of human septal chondrocytes cultured in a bioreactor are enhanced with time and are analogous to beads cultured under static culture.Prospective, basic science.Research laboratory.Human septal chondrocytes from 9 donors were expanded in monolayer and seeded in alginate beads. The beads were cultured in a rotary bioreactor for 21 days in media supplemented with growth factors and human serum, using static culture as the control. Biochemical and biomechanical properties of the beads were measured.Glycosaminoglycan (GAG) accumulation significantly increased during 2 measured time intervals, 0 to 21 days and 10 to 21 days (P.01). No significant difference was seen between the static and bioreactor conditions. Substantial type II collagen production was demonstrated in the beads terminated at day 21 of culture in both conditions. In addition, the biomechanical properties of the beads were significantly improved at 21 days in comparison to beads from day 0.Human septal chondrocytes cultured in alginate beads exhibit significant matrix deposition and improved biomechanical properties after 21 days. Alginate bead diameter and stiffness positively correlated with GAG and type II collagen accretion. Matrix production in beads is supported by the use of a rotary bioreactor.

In the CNS of leech, the central projections of peripheral sensory neurons segregate into three distinct axonal tracts during early development. We have previously shown that a subset of these neurons, recognized by the monoclonal antibody lan 4-2, projects axons into only one of these fascicles (Johansen et al., 1992, Neuron 8, 599). Here we report on a developmental and biochemical characterization of another fascicle-specific antigen labeled by the monoclonal antibody lan 3-6. By immunocytochemistry and double labelings we demonstrate that the lan 3-6 epitope is expressed only by a small subgroup of the peripheral neurons in Macrobdella embryos. The axons of these neurons selectively fasciculate in the CNS, but to only one of the three lan 3-2-positive tracts, which is different from the previously described lan 4-2-positive tract. These observations support the existence of a hierarchy of guidance cues mediating specific tract formation in this system. A biochemical analysis of the antigen suggests that it is likely to be a glycosylated protein with a molecular weight of approximately 200 kDa. Thus, the restricted expression of the lan 3-6 antigen and its biochemical properties are consistent with the hypothesis that this antigen may be playing a role in axonal guidance.