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: To produce autologous human septal neocartilage constructs substantially larger in size than previously produced constructsTo demonstrate that volume expanded neocartilage constructs possess comparable histological and biochemical properties to standard size constructsTo 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. MAIN OUTCOMES MEASURED: 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, histological, 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. Histological 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 constructs. Additionally, glycosaminoglycan (GAG) and total collagen content did not significantly differ between the two construct sizes. Biomechanical analysis of the 24mm and 12mm constructs demonstrated comparable compressive and tensile properties. CONCLUSION AND RELEVANCE: Volume expanded human septal neocartilage constructs are qualitatively and histologically similar to standard 12mm constructs. Biochemical and biomechanical analysis of the constructs demonstrated equivalent properties. This study shows that modification of existing protocols is not required to successfully produce neocartilage constructs in larger sizes for reconstruction of more substantial craniofacial defects. LEVEL OF EVIDENCE: NA.

Shape fidelity of native and engineered human nasal septal cartilage.

OBJECTIVE: 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. STUDY DESIGN: Prospective, basic science. SETTING: Laboratory. SUBJECTS AND METHODS: 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. RESULTS: 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. CONCLUSION: 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.

Flexural properties of native and tissue-engineered human septal cartilage.

OBJECTIVE: To determine and compare the bending moduli of native and engineered human septal cartilage. STUDY DESIGN: Prospective, basic science. SETTING: Research laboratory. SUBJECTS AND METHODS: Neocartilage 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. RESULTS: All 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). CONCLUSION: Human 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 maturation relative to native tissue.

Culture of human septal chondrocytes in a rotary bioreactor.

OBJECTIVES: (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. STUDY DESIGN: Prospective, basic science. SETTING: Research laboratory. METHODS: 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. RESULTS: 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. CONCLUSION: 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 vivo implantation of tissue-engineered human nasal septal neocartilage constructs: a pilot study.

OBJECTIVE: To determine the in vivo biocompatibility of septal neocartilage constructs developed in vitro by an alginate intermediate step. STUDY DESIGN: Prospective, animal model. SETTING: Research laboratory. SUBJECTS AND METHODS: A murine model was used to examine the maturation of neocartilage constructs in vivo. Chondrocytes collected from patients undergoing septoplasty were expanded in monolayer and suspended in alginate beads for 3-dimensional culture in media containing human serum and growth factors. After in vitro incubation for 5 weeks, the constructs were implanted in the dorsum of athymic mice for 30 and 60 days (n = 9). After the mice were sacrificed, the constructs were recovered for assessment of their morphological, histochemical, biochemical, and biomechanical properties. RESULTS: The mice survived and tolerated the implants well. Infection and extrusion were not observed. Neocartilage constructs maintained their general shape and size and demonstrated cell viability after implantation. The implanted constructs were firm and opaque, sharing closer semblance to native septal tissue relative to the gelatinous, translucent preimplant constructs. Histochemical staining with hematoxylin and eosin (H&E) revealed that the constructs exhibited distinct morphologies characteristic of native tissue, which were not observed in preimplant constructs. DNA and type II collagen increased with duration of implantation, whereas type I collagen and glycoaminoglycans (GAG) decreased. Mechanical testing of a 60-day implanted construct demonstrated characteristics similar to native human septal cartilage. CONCLUSIONS: Neocartilage constructs are viable in an in vivo murine model. The histologic, biochemical, and biomechanical features of implanted constructs closely resemble native septal tissue when compared with preimplant constructs.