Insulin-like growth factor-I and growth differentiation factor-5 promote the formation of tissue-engineered human nasal septal cartilage.

INTRODUCTION: Tissue engineering of human nasal septal chondrocytes offers the potential to create large quantities of autologous material for use in reconstructive surgery of the head and neck. Culture with recombinant human growth factors may improve the biochemical and biomechanical properties of engineered tissue. The objectives of this study were to (1) perform a high-throughput screen to assess multiple combinations of growth factors and (2) perform more detailed testing of candidates identified in part I. METHODS: In part I, human nasal septal chondrocytes from three donors were expanded in monolayer with pooled human serum (HS). Cells were then embedded in alginate beads for 2 weeks of culture in medium supplemented with 2% or 10% HS and 1 of 90 different growth factor combinations. Combinations of insulin-like growth factor-I (IGF-1), bone morphogenetic protein (BMP)-2, BMP-7, BMP-13, growth differentiation factor-5 (GDF-5), transforming growth factor ß (TGFß)-2, insulin, and dexamethasone were evaluated. Glycosaminoglycan (GAG) accumulation was measured. A combination of IGF-1 and GDF-5 was selected for further testing based on the results of part I. Chondrocytes from four donors underwent expansion followed by three-dimensional alginate culture for 2 weeks in medium supplemented with 2% or 10% HS with or without IGF-1 and GDF-5. Chondrocytes and their associated matrix were then recovered and cultured for 4 weeks in 12 mm transwells in medium supplemented with 2% or 10% HS with or without IGF-1 and GDF-5 (the same medium used for alginate culture). Biochemical and biomechanical properties of the neocartilage were measured. RESULTS: In part I, GAG accumulation was highest for growth factor combinations including both IGF-1 and GDF-5. In part II, the addition of IGF-1 and GDF-5 to 2% HS resulted in a 12-fold increase in construct thickness compared with 2% HS alone (p < 0.0001). GAG and type II collagen accumulation was significantly higher with IGF-1 and GDF-5. Confined compression modulus was greatest with 2% HS, IGF-1, and GDF-5. CONCLUSION: Supplementation of medium with IGF-1 and GDF-5 during creation of neocartilage constructs results in increased accumulation of GAG and type II collagen and improved biomechanical properties compared with constructs created without the growth factors.

Growth of human septal chondrocytes in fibrin scaffolds.

BACKGROUND: Tissue engineering of nasal septal cartilage has been the focus of research owing to its superior structural rigidity and ease of harvest. In vitro constructs formed from septal chondrocytes using fibrin glue within a polyglycolic acid (PGA) scaffold have been shown to be viable, but their cellular growth and expression of differentiated features still have not been quantified. In this study, we evaluated cellular proliferation and production of cartilaginous extracellular matrix (ECM) components in fibrin glue preparations within a PGA scaffold. METHODS: Human chondrocytes were expanded for one passage in monolayer in culture medium. The cells were then grown in (1) fibrinogen, (1/2)x-thrombin, (1/2)x (F/2:T/2); (2) fibrinogen, 1/10x-thrombin, 1/10x (F/10:T/10); (3) fibrinogen, 1x-thrombin, 1/100x (F/1:T/100). RESULTS: Cellular proliferation and glycosaminoglycan (GAG) production per cell were highest in the F/2:T/2 preparations. Greater proliferation was seen in chondrocyte-fibrin composites seeded onto the PGA scaffold when compared with chondrocytes seeded onto the PGA scaffold alone. No significant difference in GAG production was seen. CONCLUSION: The addition of fibrin glue to chondrocytes seeded onto a PGA scaffold results in increased cellular proliferation while maintaining production of ECM components. Long-term stable fibrin gels in combination with PGA scaffolds may facilitate generation of cartilaginous tissue for use in reconstructive surgery.

Cartilage outgrowth in fibrin scaffolds.

BACKGROUND: Fibrin glue has been a favorable hydrogel in cartilage tissue engineering, but implantation of chondrocyte-fibrin suspensions have resulted in volume loss. In this study, human septal cartilage chips were seeded onto a fibrin scaffold, and cellular proliferation and production of cartilaginous extracellular matrix (ECM) were evaluated. METHODS: Human septal cartilage was diced into cartilage chips and encased with and without fibrin glue. Four conditions were initially tested for DNA content and glycosaminoglycan (GAG) production: (1) control medium in tissue culture, (2) control medium with fibrin glue, (3) collagenase-supplemented medium in tissue culture, and (4) collagenase-supplemented medium seeded in fibrin glue. Cartilage chips cultured in collagenase-treated medium were then seeded onto either cell culture plates, suspended in alginate, or mixed with fibrin. Cellular proliferation, GAG production, and histochemistry were evaluated. RESULTS: Fibrin preparations increased cellular proliferation and DNA content. GAG levels were highest in collagenase-treated samples encased in fibrin. Cartilage chips treated with collagenase showed increased cellular proliferation in the fibrin preparations compared with preparations without fibrin. GAG increased with the addition of fibrin when compared with explant. Histochemistry revealed increased GAG accumulation in the regions between the cartilage chips with the addition of fibrin. CONCLUSION: Adding fibrin glue to collagenase-treated cartilage chips results in increased proliferation and maintains ECM production and, therefore, may facilitate generation of cartilaginous tissue for use in reconstructive surgery.

Effect of bone morphogenetic proteins 2 and 7 on septal chondrocytes in alginate.

OBJECTIVE: To determine the effects of bone morphogenetic proteins (BMP)-2 and -7, and serum, on extracellular matrix production by human septal chondrocytes in alginate. STUDY DESIGN: Human nasal septal chondrocytes were expanded, suspended in alginate, and cultured in BMP-2 or 7, with and without serum. The optimal concentration of each growth factor was determined based on matrix production. Next, the synergistic effects of BMP-2 and -7 at optimal concentrations were determined on separate beads, based on matrix quantity and histology. RESULTS: Matrix content was highest with concentrations of BMP-2 and -7 of 100 ng/ml and 20 ng/ml, respectively, with serum. Adding both BMP-2 and -7, with serum, increased matrix content by factors of 5.1 versus serum-only cultures, 2.7 versus only BMP-2 with serum, and 2.4 versus only BMP-7 with serum. All comparisons were statistically significant. CONCLUSION: BMP-2 and -7 significantly increase production of extracellular matrix by septal chondrocytes suspended in alginate. The presence of serum improves matrix production. SIGNIFICANCE: BMP-2 and -7 have great potential for use in cartilage tissue engineering.

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, HAO, 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, HAO, 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 kp at 15% offset compression (p = 0.12) and a borderline significant effect of orientation on kp 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 HAO, 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.

Stored human septal chondrocyte viability analyzed by confocal microscopy.

OBJECTIVES: To analyze the effects of prolonged storage time, at warm and cold temperatures, on the viability of human nasal septal chondrocytes and to understand the implications for tissue engineering of septal cartilage. DESIGN: Basic science. SUBJECTS: Septal cartilage was obtained from 10 patients and placed in bacteriostatic isotonic sodium chloride solution. Four specimens were kept at 23 degrees C, and 4 were kept at 4 degrees C. The viability of the chondrocytes within the cartilage was assessed using confocal laser scanning microscopy every 5 days. The 2 other specimens were assessed for viability on the day of harvest. RESULTS: Viability on the day of harvest was 96%, implying minimal cell death from surgical trauma. After 1 week, cell survival in all specimens was essentially unchanged from the day of harvest. At 23 degrees C, the majority (54%) of cells were alive after 20 days. At 4 degrees C, 70% of cells survived 1 month and 38% were alive at 2 months. Qualitatively, chondrocytes died in a topographically uniform distribution in warm specimens, whereas cold specimens displayed a more irregular pattern of cell death. CONCLUSION: Septal chondrocytes remain viable for prolonged periods when stored in simple bacteriostatic isotonic sodium chloride solution, and such survival is enhanced by cold storage.

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.

Growth and phenotype of low-density nasal septal chondrocyte monolayers.

OBJECTIVE: To analyze the growth patterns and differentiation of human septal chondrocyte monolayers of different seeding densities. STUDY DESIGN: Chondrocytes from 8 donors were plated at densities ranging from 20,000 cells/cm(2) (high density) to 300 cells/cm(2) (very low density). Confluency, cellularity, and glycosaminoglycan content were determined from days 1 to 15. RESULTS: Confluency was attained at 5.8, 8.3, 11.0, and 14.8 days for high-, intermediate-, low-, and very low-density monolayers, respectively (P < 0.001). Regression growth curves showed typical lag, logarithmic, and stationary phases. Confluent monolayers attained similar cellularity (power = 0.94) and differentiation (power = 0.88), regardless of initial density. CONCLUSIONS: Human septal chondrocyte monolayers reach confluency from very low initial densities. Growth patterns, cellularity, and differentiation are similar to other starting densities. SIGNIFICANCE: Very low-density monolayers expanded cell number 838-fold in 1 passage and therefore are sufficient for tissue-engineering purposes. This is important because of the requirement of maintaining differentiation and the limitation of small tissue harvest specimens.

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 patient's 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. STUDY DESIGN: Prospective, randomized, controlled study. METHODS: Nasal septal chondrocytes from six patient donors were isolated by enzymatic digestion and expanded in monolayer culture in both serum-free media (SFM) and 2% fetal bovine serum (FBS). Both of these groups were exposed to varying concentrations and combinations of transforming growth factor (TGF)-beta1, basic fibroblast growth factor (FGF)-2 both at 1, 5, and 25 ng/mL, and bone morphogenetic protein (BMP)-2 and insulin-like growth factor (IGF)-1, both at 5, 25, and 125 ng/mL in the medium during the expansion phase. Cell morphology was assessed throughout the culture duration. After 7 days of monolayer growth, cultures were assessed for cellularity and glycosaminoglycan (GAG) content. RESULTS: The addition of low-dose FBS in culture media consistently led to significantly greater cell proliferation and matrix deposition than the SFM cell cultures. FGF-2 and TGF-beta1 both alone and in combination led to the greatest proliferative effect compared with the other growth factors. In contrast, BMP-2 and IGF-1 led to the least cell proliferation although was most effective in retaining chondrocyte cell morphology. CONCLUSIONS: With the addition of TGF-beta1 and FGF-2 to culture media, the concentration of serum can be greatly decreased and possibly eliminated altogether without jeopardizing cell proliferation.

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).