Deborah Watson

Human Septal Chondrocyte Redifferentiation in Alginate, Polyglycolic Acid Scaffold, and Monolayer Culture†

Objectives/Hypothesis Tissue engineering laboratories are attempting to create neocartilage that could serve as an implant material for structural support during reconstructive surgery. One approach to forming such tissue is to proliferate chondrocytes in monolayer culture and then seed the expanded cell population onto biodegradable scaffolds. However, chondrocytes are known to dedifferentiate after this type of monolayer growth and, as a result, decrease their production of cartilaginous extracellular matrix components such as sulfated glycosaminoglycans. The resultant tissue lacks the biomechanical properties characteristic of cartilage. The objective of the study was to determine whether different culture systems could induce monolayer‐expanded human septal chondrocytes to redifferentiate and form extracellular matrix.

Inverting papilloma of the head and neck: the UCLA update.

Inverting papilloma of the nose and paranasal sinuses is a benign disease with malignant potential. This disease is characterized by multiple recurrences, especially after minimal operative therapy. Controversy exists over the most appropriate treatment for this rare tumor. This review presents an update of the UCLA experience with inverting papilloma over the past four decades along with a review of the literature. A retrospective study of 61 patients seen at the UCLA Medical Center was conducted. The mean age at presentation was 63 years, with a male-to-female ratio of 2:1. The most common symptom at presentation was nasal obstruction (71%), followed by epistaxis (27%). Seventeen percent of the patients in this series either had concurrent squamous cell carcinoma of the nose or paranasal sinuses, or it developed. Patients treated with a lateral rhinotomy and medial maxillectomy had a recurrence rate of 30 percent. Those treated with a less aggressive operation had a recurrence rate of 71 percent. Despite a trend for a more conservative sinus operation in recent literature, we continue to advocate a lateral rhinotomy and medial maxillectomy as the treatment of choice for inverting papilloma of the head and neck.

Effects of Serial Expansion of Septal Chondrocytes on Tissue-Engineered Neocartilage Composition

OBJECTIVES: Cartilage grafts for reconstructive surgery may someday be created from harvested autologous chondrocytes that are expanded and seeded onto biodegradable scaffolds in vitro. This study sought to quantify the biochemical composition of neocartilage engineered from human septal chondrocytes and to examine the effects of cell multiplication in monolayer culture on the ultimate composition of the neocartilage. METHODS: Human septal chondrocytes from 10 donors were either seeded immediately after harvest (passage 0 (P0)) onto polyglycolic acid (PGA) scaffolds or underwent multiplication in monolayer culture before scaffold seeding at passage 1 (P1) and passage 2 (P2). Cell/scaffold constructs were grown in vitro for 7, 14, and 28 days. Neocartilage constructs underwent histologic analysis for matrix sulfated glycosaminoglycan (S-GAG) and type II collagen as well as quantitative assessment of cellularity (Hoescht 33258 assay), S-GAG content (dimethylmethylene blue assay), and collagen content (hydroxyproline assay). RESULTS: Histologic sections of constructs seeded with P0 cells stained strongly for S-GAG and type II collagen, whereas decreased staining for both matrix components was observed in constructs derived from P1 and P2 cells. Cellularity, S-GAG content, and total collagen content of constructs increased significantly from day 7 to day 28. S-GAG accumulation in P0 constructs was higher than in either P1 (P < 0.05) or P2 (P < 0.01) constructs, whereas cellularity and total collagen content showed no difference between passages. CONCLUSION: Neocartilage created from chondrocytes that have undergone serial passages in monolayer culture exhibited decreased matrix S-GAG and type II collagen, indicative of cellular dedifferentiation. SIGNIFICANCE: The alterations of matrix composition produced by dedifferentiated chondrocytes may limit the mechanical stability of neocartilage constructs. The process of tissue engineering of cartilage was introduced by Vacanti et al 1,2 more than a decade ago as a potential solution to the limited supply of cartilage autografts available for reconstructive surgery. One strategy of tissue engineering is initiated by cartilage harvest from a donor site such as the nasal septum or the auricle. After digestion of the cartilage extracellular matrix, the chondrocytes can be isolated and grown in vitro using standard cell culture methods. Cell populations are expanded in culture to yield large numbers of chondrocytes. These cells can then be seeded onto biodegradable scaffolds and induced to deposit an extracellular matrix, thus forming new cartilage. Such neocartilage could potentially be implanted for structural support during reconstructive surgical procedures. There are several potential advantages of using tissue-engineered cartilage over native cartilage for autografting. First, the ability of chondrocytes to replicate in vitro allows for the expansion of cell numbers to produce theoretically limitless supplies of cartilage autografts. Furthermore, by varying the geometric configurations of the scaffolds, neocartilage autografts could potentially be designed in any desired size and shape. Finally, neocartilage constructs derived from autologous chondrocytes have a lower risk of immune rejection and infection transmission than that encountered with cartilage allografts or xenografts. Although a variety of scaffold materials exist, much research in tissue engineering has focused on scaffolds consisting of a nonwoven mesh of bioresorbable fibers such as polyglycolic acid (PGA), polylactic acid, or their copolymer. 3 The interlacing scaffold fibers provide a 3-dimensional structure to which cells can adhere. Chondrocytes grown in these scaffolds deposit extracellular matrix around themselves that is remodeled as the synthetic fibers degrade, theoretically creating cartilaginous tissue in the shape of the original scaffold. Chondrocytes for tissue engineering research have been obtained from a variety of sources, including articular, costal, nasal, and auricular cartilage from both animals and humans. 4–9 Septal cartilage can be harvested with less morbidity than articular or costal cartilage and has superior mechanical stability compared with elastic cartilage from the ear. Septal chondrocytes might therefore be used to form neocartilage that replicates the mechanical properties of native septal cartilage. Despite these advantages, limited studies have used septal chondrocytes for neocartilage formation on biodegradable scaffolds. 8–10 These studies have used histologic architecture as an outcome measure of successful neocartilage creation on scaffolds. Indeed, these authors have demonstrated the production of neocartilage that histologically resembles native cartilage. 8–10 When reimplanted into animal models, however, neocartilage constructs have uniformly lacked long-term structural integrity, demonstrating invasion by fibroblasts and loss of shape over time. 6,11 Unknown differences in the composition of neocartilage compared with native cartilage may be responsible for these phenomena. Native cartilage is composed of nests of cells embedded in an extensive extracellular matrix consisting of large proteoglycan molecules interwoven with collagen fibrils. Proteoglycans are macromolecules consisting of a protein core with hundreds of sulfated glycosaminoglycan side chains. These glycosaminoglycan chains consist of repeating disaccharide units whose highly negative charge attracts osmotically active cations. The osmotic ingression of water confers turgor to the tissue, which allows the cartilage to resist compressive forces. Collagen fibrils in the extracellular matrix serve a complementary mechanical function by resisting tensile forces. In hyaline cartilage, type II collagen predominates over other collagen subtypes, whereas the matrix of other connective tissues (skin, tendon, bone, ligaments) is primarily composed of type I collagen. Because the mechanical properties of cartilage are largely due to the composition and structure of its extracellular matrix, it is possible that a deficient matrix composition plays a role in the lack of neocartilage integrity to date. In support of this hypothesis is the observation that neocartilage engineered on scaffolds from calf articular or costal chondrocytes demonstrated less matrix glycosaminoglycan than native articular cartilage. 4,5 Thus far, no published reports have quantified the biochemical constituents of neocartilage constructed from human septal chondrocytes on biodegradable scaffolds. The distinction between data obtained from calf articular cells and adult human septal chondrocytes is important. It is unclear whether cells from different species will behave similarly under equivalent culture conditions. Furthermore, chondrocytes obtained from different anatomic locations may have different properties owing to the vastly different function served by cartilage in different areas (eg, load bearing in articular cartilage, rigid structural support for septal cartilage, and the deformation with elastic recoil characteristic of auricular cartilage). Finally, cells would be expected to grow and produce matrix in a manner that is dependent on donor maturity and age. Results from previous experimental work 12,13 have demonstrated an age-dependent decline in the synthesis of extracellular matrix components by cultured chondrocytes, suggesting that cells from juvenile subjects may have superior capacity for regenerating cartilage. Cells from younger subjects also are likely to expand in number faster and retain their chondrocyte phenotype for longer periods ex vivo. Thus, the translation of experimental data from fetal or juvenile animal chondrocytes to adult human septal cells is not straightforward. In addition, previous work has not routinely addressed another important issue relating to the practical application of cartilage engineering. As stated previously, one major advantage of tissue engineering is the potential ability to produce greater amounts of cartilage than are available from harvest during traditional autografting. This requires the expansion of cell numbers in culture, which potentially induces the chondrocytes to dedifferentiate. It is well established that with increasing passage, cultured chondrocytes exhibit a progressive transformation toward a more fibroblastic phenotype. 14 Thus, the effect on matrix formation of the necessary and potentially detrimental step of chondrocyte expansion remains to be established. The objective of the current study was to quantify the accumulation of the major cartilage matrix constituents in neocartilage engineered from adult human nasal septal chondrocytes. Additionally, the variation of the composition of neocartilage constructs from cell populations after expansion of cell numbers through multiple passages in culture was examined.

Tissue-Engineered Human Nasal Septal Cartilage Using the Alginate-Recovered-Chondrocyte Method†

Objectives Tissue engineering of nasal septal cartilage has numerous potential applications in craniofacial reconstruction. Chondrocytes suspended in alginate gel have been shown to produce a substantial cell‐associated matrix. The objective of this study was to determine whether cartilage tissue could be generated using the alginate‐recovered‐chondrocyte (ARC) method, in which chondrocytes are cultured in alginate as an intermediate step in tissue fabrication.

Effect of growth factors on cell proliferation, matrix deposition, and morphology of human nasal septal chondrocytes cultured in monolayer.

Objectives: Tissue engineering of septal cartilage provides ex vivo growth of cartilage from a patients own septal chondrocytes for use in craniofacial reconstruction. To become clinically applicable, it is necessary to rapidly expand a limited population of donor chondrocytes and then stimulate the production of extracellular matrix on a biocompatible scaffold. The objective of this study was to determine favorable serum‐free culture conditions for proliferation of human septal chondrocytes using various concentrations and combinations of four growth factors.

Tensile biomechanical properties of human nasal septal cartilage.

Background The biomechanical properties of human septal cartilage have yet to be fully defined and thereby limits our ability to compare tissue-engineered constructs to native tissue. In this study, we analyzed the tensile properties of human nasal septal cartilage with respect to axis of tension, age group, and gender. Methods Fifty-five tensile tests were run on human septal specimens obtained from 28 patients. Samples obtained in the vertical and anterior–posterior (both above and within the maxillary crest) axes were subjected to equilibrium and dynamic tensile testing. Results The average values for strength, failure strain, equilibrium modulus and dynamic modulus were not found to be significantly different with respect to axis of tension testing, age group, or gender. Tensile results for septal cartilage were as follows: equilibrium modulus 3.01 ± 0.39 MPa, dynamic modulus 4.99 ± 0.49 MPa, strength 1.90 ± 0.24 MPa, and failure strain 0.35 ± 0.03 mm/mm. Conclusion We confirm that septal cartilage has weaker tensile properties compared to articular cartilage and found no difference in strength with respect to age, gender, or axis of tension (isotropic).

Surgical Management of the Septal Perforation

Initial management of a septal perforation involves medical intervention, but there are several surgical options available. Deciding to proceed with a surgical repair is dependent on the etiology of the defect, how the symptoms impact the patient, the extent of damage or impending destruction to the nasal support, and the absence of any active disease process. The literature describes several methods for septal perforation repair; each has its technical challenges because of the tenuous nature of the tissues and limited surgical exposure of the area. This article reviews the diagnostic work-up of septal perforations, the medical management, and the surgical treatment options, with emphasis placed on the open rhinoplasty approach.

Human serum for tissue engineering of human nasal septal cartilage

Objective To compare the chondrogenic and proliferative effects of pooled human serum (HS) and fetal bovine serum (FBS) on tissue-engineered human nasal septal chondrocytes. Study Design and Setting Human chondrocytes were expanded for one passage in monolayer in medium supplemented with 10% FBS, 2% HS, 10% HS, or 20% HS. Cells were then suspended in alginate beads for 3D culture for 2 weeks with 10% FBS, 2% HS, 10% HS, or 20% HS. Results Monolayer cell yields were greater with HS than FBS. In alginate, cellular proliferation, glycosaminoglycan production per cell, and type II collagen were significantly higher with 10% HS compared to 10% FBS controls. Conclusion HS results in increased proliferation and production of cartilaginous extracellular matrix by tissue-engineered human nasal septal chondrocytes, compared to FBS controls. Significance Culture with human serum may facilitate creation of neocartilage constructs that more closely resemble native tissue.

Compressive biomechanical properties of human nasal septal cartilage.

Background Nasal septal cartilage is frequently used in nasal reconstruction and is a common source of chondrocytes for cartilage tissue engineering. The biomechanical properties of septal cartilage have yet to be fully defined and this limits the ability to compare it to the various alternative tissue-implant materials or tissue-engineered neocartilage. Given the unique structure and orientation of the septum within the nose, we sought to investigate anisotropic behaviors of septal cartilage in compression and correlate this to the concentration of glycosaminoglycans (GAG) and collagen within the cartilage. Methods Human nasal septal cartilage specimens were tested in confined compression, with each sample analyzed in a medial orientation and also either a vertical or caudal-cephalic orientation, with the order of tests randomized. The equilibrium confined compression (aggregate) modulus, HA0 and the permeability, kp, at different offset compression levels were obtained for each compression test. After testing, the cartilage samples were solubilized, and the concentrations of GAG and collagen were obtained. Results Forty-nine compression tests (24 medial, 12 vertical, 13 caudal-cephalic) were run on cartilage specimens obtained from 21 patients. There was a significant effect of orientation on compression modulus, HA0, with the vertical (0.7 ± 0.12 MPa) and caudal-cephalic (0.66 ± 0.01 MPa) orientations being significantly stiffer (p = 0.05) than the medial orientation (0.44 ± 0.04 MPa). There was a trend of an orientation effect on k at 15% offset compression (p = 0.12) and a borderline significant effect of orientation on k at 30% offset compression (p = 0.05), demonstrating the M orientation to be more permeable than both the vertical and caudal–cephalic orientations. Both univariate and multivariate analysis did not demonstrate a significant effect of order of compression, age, gender, thickness, dry/wet weight, GAG, or collagen on either HA0, or kp values (p > 0.05). Conclusion This study provides new information on the compressive properties of septal cartilage along different axes of compression. The results demonstrate that human septal cartilage is anisotropic; the compressive stiffness is higher in the vertical and caudal–cephalic orientations than in the medial orientation. Additionally, the medial orientation tends to have the greatest permeability. The data obtained in this study provide a reference to which various craniofacial reconstruction materials and tissue-engineered neocartilage can be compared.

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.