August B. Sage

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).

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.

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.

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.

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/cm2 (high density) to 300 cells/cm2 (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.

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×–thrombin, 1/2× (F/2:T/2); (2) fibrinogen, 1/10×–thrombin, 1/10× (F/10:T/10); (3) fibrinogen, 1×–thrombin, 1/100× (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. Conclusions 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.