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Figure 1.
Buckled construct with a smooth yellowish gray surface and rounded corners during solid-body rotation in the slowly turning lateral vessel. This construct was seeded with 9.6 × 106 cells.

Buckled construct with a smooth yellowish gray surface and rounded corners during solid-body rotation in the slowly turning lateral vessel. This construct was seeded with 9.6 × 106 cells.

Figure 2.
Construct seeded with 5.5×106 cells in serum-free medium seen after 72 hours. Uniform seeding and basophilic matrix, in addition to a cluster of developing hyaline cartilage harboring pyknotic nuclei, are evident (hematoxylin-eosin, original magnification ×20).

Construct seeded with 5.5×106 cells in serum-free medium seen after 72 hours. Uniform seeding and basophilic matrix, in addition to a cluster of developing hyaline cartilage harboring pyknotic nuclei, are evident (hematoxylin-eosin, original magnification ×20).

Figure 3.
Construct seeded with 2.3×106 cells seen after 34 days in the spinner flask. A gradient of matrix formation from the peripheral zones of the construct to the more central zones is apparent (hematoxylin-eosin, original magnification ×10).

Construct seeded with 2.3×106 cells seen after 34 days in the spinner flask. A gradient of matrix formation from the peripheral zones of the construct to the more central zones is apparent (hematoxylin-eosin, original magnification ×10).

Figure 4.
Section of a construct seeded with 9.6×106 cells after 5 days in the spinner flask. An abundance of immature matrix and clusters of more mature-looking hyaline cartilage are evident (Masson trichrome, original magnification ×20).

Section of a construct seeded with 9.6×106 cells after 5 days in the spinner flask. An abundance of immature matrix and clusters of more mature-looking hyaline cartilage are evident (Masson trichrome, original magnification ×20).

Figure 5.
Section of a construct after 12 days in the slowly turning lateral vessel (ie, total cultivation time of 17 days). An abundance of friable-appearing and less ground substance, like staining characteristic of immature matrix, is observed among the polymer fragments (safronin-O, original magnification ×10).

Section of a construct after 12 days in the slowly turning lateral vessel (ie, total cultivation time of 17 days). An abundance of friable-appearing and less ground substance, like staining characteristic of immature matrix, is observed among the polymer fragments (safronin-O, original magnification ×10).

Figure 6.
Section of a construct seeded with cryogenically preserved chondrocytes at 8.6×106 cells per scaffold. Uniformity of seeding and a spectrum of matrix generation, including clusters of hyaline cartilage with round chondrocytes in lipid-filled lacunae, are evident (Masson trichrome, original magnification ×20).

Section of a construct seeded with cryogenically preserved chondrocytes at 8.6×106 cells per scaffold. Uniformity of seeding and a spectrum of matrix generation, including clusters of hyaline cartilage with round chondrocytes in lipid-filled lacunae, are evident (Masson trichrome, original magnification ×20).

Figure 7.
Kinetics of seeding. Data for serum free (A), control (C), 2 scaffolds (D), 1 scaffold (E), frozen chondrocytes (F), and 3 scaffolds (H) are shown.

Kinetics of seeding. Data for serum free (A), control (C), 2 scaffolds (D), 1 scaffold (E), frozen chondrocytes (F), and 3 scaffolds (H) are shown.

Figure 8.
Scanning electron micrograph of a 5-day construct after seeding in the spinner flask at 6.4×106 cells per scaffold (original magnification ×80). Adherent chondrocytes appear in the process of filling the void spaces with de novo extracellular matrix material.

Scanning electron micrograph of a 5-day construct after seeding in the spinner flask at 6.4×106 cells per scaffold (original magnification ×80). Adherent chondrocytes appear in the process of filling the void spaces with de novo extracellular matrix material.

Seeding Kinetics Experiments*
Seeding Kinetics Experiments*
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Bujia  JSittinger  MMinuth  WWHammer  CBurmester  GKastenbauer  E Engineering of cartilage tissue using bioresorbable polymer fleeces and perfusion culture. Acta Otolaryngol (Stockh). 1995;115307- 310Article
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Naumann  ARotter  NBujia  JAigner  J Tissue engineering of autologous cartilage transplants for rhinology. Am J Rhinol. 1998;1259- 63Article
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Peulacher  WCMooney  DLanger  RUpton  JVacanti  JPVacanti  CA Design of nasoseptal cartilage replacements synthesized from biodegradable polymers and chondrocytes. Biomaterials. 1994;15774- 778Article
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Original Article
June 2000

Growth of Tissue-Engineered Human Nasoseptal Cartilage in Simulated Microgravity

Author Affiliations

From the Wound Healing and Tissue Engineering Laboratory, Division of Otolaryngology–Head and Neck Surgery, Stanford University, Stanford, Calif.

Arch Otolaryngol Head Neck Surg. 2000;126(6):759-765. doi:10.1001/archotol.126.6.759
Abstract

Objective  To evaluate the feasibility of in vitro fabrication of tissue-engineered cartilage from human nasoseptal chondrocytes for autologous reconstruction.

Design  Hyaline cartilage was reconstituted from chondrocyte–polyglycolic acid scaffolding constructs in a 3-dimensional mammalian cell culture cascade. This included monolayer cellular amplification, cell seeding in the spinner flask, and tissue growth in simulated microgravity.

Results  The quality of the fabricated cartilage analogue was found to depend on the initial cell density, duration of incubation, and bioreactor type. Dynamic seeding was nearly completed within the first 10 hours of inoculation regardless of the cell source (cryogenically preserved vs fresh chondrocytes) or presence of serum. A duration of incubation in excess of 4 weeks was required for complete matrix biosynthesis at low seeding densities in the spinner flasks. Seeding densities greater than 2.3 × 106 chondrocytes per scaffold were required for early hyaline cartilage formation as well as longer-time mature matrix regeneration. In addition, maintaining the structural integrity of the unreinforced scaffold, which is necessary for continued mature matrix regeneration, was achievable through postseeding tissue growth in simulated microgravity.

Conclusion  Once combined with polyglycolic acid scaffolds in the bioreactor cascades that allow efficient seeding and quiescent tissue growth, human septal chondrocytes become a valuable source of reproducible ex vivo cartilage regeneration in the laboratory.

TISSUE ENGINEERING, based on the use of Food and Drug Administration–approved synthetic biodegradable polymer cell delivery vehicles (scaffolds), was introduced in 1988 by Vacanti et al1 as an alternate treatment for replacing missing cartilage. By virtue of ability to maintain their original 3-dimensional shape, homopolymers of polyglycolic acid (PGA) were configured into branching networks of thin fibers, coated with a solvent-based poly-L-lactic acid suspension, molded into the shape of a human ear, impregnated with a large population of bovine-derived articular chondrocytes, and implanted subcutaneously on the dorsum of athymic mice. These cell-polymer constructs reportedly generated cartilage analogues that approximated the biochemical and biomechanical properties of articular cartilage in 6 and 12 weeks of in vivo time, respectively. Numerous more recent animal-based in vivo2 and in vitro3 studies based on 3-dimensional cell culture paradigms predict that tailor-made cartilage substitutes for craniofacial augmentation,4 joint resurfacing,5 and tracheal replacement6 are not too far from clinical realization.

Although still in early stages of development, tissue engineering is beginning to provide a promising means of replacing irreversibly lost tissue for millions of individuals worldwide on an annual basis.7 A number of reports centering on the in vivo application of tissue engineering have appeared in the literature. However, the majority of these investigations have focused on xenotransplantation of in vitro regenerated articular or costal chondrocyte-polymer constructs into animal recipients, with unpredictable degrees of success.8 For example, the athymic mouse model, which has been the focus of the majority of the pioneering in vivo tissue engineering investigations, is limited in terms of ability to predict the outcome in an immunologically competent animal. In vivo animal models have also suffered from lack of reproducibility, as demonstrated by conflicting results in rabbit vs canine studies.9 While chondrocyte infusion into a defect in rabbits resulted in observable reparative activity, the canine models failed to show any such benefit, potentially because of age-specific and species-specific characteristics of articular chondrocytes. More recently, autologous in vivo transplantation in a rabbit model system has produced poor results that are most likely due to lack of in vitro optimization before implantation.10 Because of these in vivo limitations, in vitro tissue engineering has been introduced as a controllable alternative for reproducible fabrication of cartilage analogues in the laboratory.

An ideal human construct biochemically, biomechanically, histologically, and morphologically approximates native cartilage and is reproducibly manufacturable in a timely fashion at a relatively low cost. In reconstructive surgery, morphometric structural constraints that are imposed by the tissue being replaced preclude the use of traditional cell culture techniques. For example in vitro regeneration of 4-mm-thick septal cartilage would require an optimal number of chondrocytes (ie, >20 million cells per cubic centimeter of scaffold) for proper induction of differentiation through enhanced cell-cell as well as cell-matrix interaction.11 However, because of diffusional constraints, these optimal cell densities are too high to allow cultivation in conventional static bacteriologic culture dishes. Because of the additional constraint imposed by the limited growth potential of fully mature adult chondrocytes, fabrication of cartilage replacements that use this type of cell source (ie, autologous chondrocytes from patient biopsy specimens) must be optimized. It is the degree of optimization in the in vitro phase that determines the quality of the engineered cartilage at implantation.

In contrast to the plethora of animal-based studies, the tissue engineering literature on human chondrocytes is sparse. In particular, only a handful of studies have focused on the ex vivo regeneration of human septal cartilage from isolated human septal chondrocytes.1215 It is generally agreed among surgeons performing head and neck reconstructive surgery that septal cartilage is the best implant to use when repairing cartilaginous defects. It has a firm nonbendable quality that gives it superior supportive properties. It is usually preferable in these characteristics to cartilage borrowed from the patient's rib or ears. It is also preferable to allografts, which can be rejected by the body. In addition, from an infectious disease point of view, it is unsafe to transplant live septal cartilage from one patient to another. The optimal situation usually entails taking some of the patient's own septal cartilage and moving it to a region where it can lend more support. Unfortunately, there is only a finite amount of septal cartilage available in a person's nose. The scope of this problem is exemplified by some surgeons who bank their patient's septal cartilage every time they operate on the nose.16

Our long-term goal is to fabricate autologous cartilage from a purely human source for implantation to repair various facial and airway defect sites. Our laboratory has already successfully expanded human septal chondrocytes in a serum-free in vitro model.17 As a prelude to in vitro fabrication of hyaline cartilage in a completely serum-free process, we studied the feasibility of ex vivo generation of cartilage tissue equivalents from fresh and cryogenically preserved septal chondrocytes. Because fabrication of implants for human use requires high-fidelity tissue regeneration, our studies combined the use of cell-PGA scaffolding constructs for efficient cell delivery with bioreactors. The latter consisted of the shear-attenuated environment of simulated microgravity, which enhanced induction of matrix formation, and a dynamic seeding phase in the magnetically stirred spinner flasks for optimal cellular attachment to the polymer surface. Emphasis was given to histological examination to determine cellularity as well as phenotypic matrix formation. Electron microscopy was used to probe tissue micromorphological characteristics and cellular attachment to the PGA fibers.

MATERIALS AND METHODS
CHONDROCYTE HARVESTING, ISOLATION, AMPLIFICATION, AND 3-DIMENSIONAL CELL CULTURE

Septal cartilage was obtained from patients during elective septoplasty. These specimens would normally be discarded, and their use has been approved by the Human Subjects Committee of the Stanford University Medical Center, Stanford, Calif. Each septal cartilage specimen was minced and digested with collagenase and deoxyribonuclease for 6 hours to isolate the chondrocytes. Fresh as well as cryogenically preserved chondrocytes18 (1-mL cell suspension in liquid nitrogen kept in dimethyl sulfoxide preservative for 6 months) were first plated in the conventional tissue culture flasks for multiplication according to the method of Klagsbrun.19 This was followed by a 24-hour period of prewetting.

Next was dynamic seeding20 of the chondrocytes onto nonwoven PGA scaffolds that were suspended from 9-cm-long spinal needles in the magnetically stirred spinner flasks at 50 rpm in chondrocyte media containing 10% fetal calf serum (FCS), 1% penicillin-streptomycin-amphotericin, 1% L-glutamine, and 12.5-mg/500 mL ascorbic acid in Dulbecco modified Eagle medium type F.12 The seeding kinetics were followed by periodic cell counts on the bulk medium with the use of the hemocytometer. One experiment was carried out in the absence of FCS. The initial seeding density varied between 2.3×106 and 43.7×106 in 6 independent experiments. The seeding densities selected were based on the lower and higher ends of those reported with the use of various animal and human chondrocyte sources. The number of scaffolds was varied between 0 and 4. As metabolic by-products accumulated and the polymer degraded, the color of the media changed from clear ruby red to turbid orange, at which point the media were exchanged. After 5 to 7 days' growth, the cell-polymer constructs either were transferred to the 110-cc slowly turning lateral vessel (STLV; Synthecon, Houston, Tex), developed by the National Aeronautics and Space Administration, for further growth up to 17 total culture days or were left in the spinner flask for a total of 34 days' growth. The rotation rate of the STLV was periodically varied to keep the constructs in solid-body rotation. Media were exchanged every 3 to 4 days. All incubations were carried out at 37°C in the presence of 5% carbon dioxide in humidified air. The scaffolds were sterilized with gaseous ethylene oxide.

CONSTRUCT ANALYSIS

The constructs were removed from the vessels at various time points for analysis. Select microtome-cut (3-8 µm thick) sections were fixed in 10% formaldehyde, embedded in paraffin, and stained with hematoxylin-eosin for cellularity and with safranin-O and Masson trichrome for the presence of chondroitin sulfates and other glycosaminoglycans. The modified tetrazolium-based colorimetric assay, as described previously by other investigators,3 was used on one construct that had been incubated in the spinner flask for 16 days to determine the presence or absence of viable cells. Briefly, the construct was placed in a microtiter well that contained 5-mg/mL thiazolyl blue under the fume hood and incubated for 4 hours. The cells were then treated with sodium dodecyl sulfate and color crystals were dissolved with isopropanol. Absorption at 560 nm was measured spectrophotometrically. Separate constructs were fixed in glutaraldehyde and gold coated for scanning electron microscopy.

RESULTS
GROSS MORPHOLOGICAL CHARACTERISTICS

Constructs that were seeded with 2.3 × 106 cells per scaffold and were cultivated in a spinner flask in the presence of FCS (250 mL) for 34 days appeared friable, soft textured, collapsible under gravity, neutrally buoyant in media, easily breakable on handling, and grayish white in gross appearance, but did not fall apart during the entire run, as previously reported in the literature.20 By contrast, chondrocyte-polymer constructs that were independently seeded at 8.1 × 106 cells per scaffold in a reduced media volume (120 mL), under otherwise identical conditions, buckled on seeding and became nearly of pastelike consistency before dissolving into media after 17 days' incubation. In contrast to constructs that were cultured in a spinner flask for 34 days, the constructs that were transferred into an STLV after 1 week or less of incubation in a spinner flask appeared to have a smooth yellowish gray surface and rounded corners (Figure 1). One of these constructs, which was seeded at 9.6 × 106 cells per scaffold, continued to remain in a buckled configuration. This buckling occurred during the spinner flask phase of cultivation before the STLV phase. By contrast, another construct that had been independently seeded at 43.7 × 106 cells per scaffold appeared more flexible as it regained its original flattened-out configuration during the STLV phase of cultivation.

HISTOLOGICAL FINDINGS

Figure 2 shows a 7-µm section of a hematoxylin-eosin–stained construct that was seeded with chondrocytes at 5.5 × 106 cells per scaffold in serum-free medium for 72 hours. Uniform seeding and basophilic matrix are evident. A cluster of developing hyaline cartilage harboring pyknotic nuclei is evident.

Figure 3 shows a 3-µm section of a hematoxylin-eosin–stained construct that was seeded with 2.3 × 106 cells per scaffold and was cultivated in a spinner flask in the presence of FCS (250 mL) for 34 days before being removed for analysis. In addition to demonstrating uniform seeding of chondrocytes among the polymer fragments, Figure 3 illustrates the gradient of matrix formation from the peripheral zones of the construct to the more central zones. The matrix zone appears rough and less basophilic than expected of fully matured ground substance.

Figure 4 represents an 8-µm section of a polymer scaffold that was seeded with chondrocytes at 9.6 × 106 cells per scaffold and cultivated in the presence of FCS in a spinner flask for 5 days before being removed for analysis. A Masson trichrome stain, which is specific for chondroitin sulfate, was used for this purpose. This figure illustrates uniform seeding of the scaffold with chondrocytes at a relatively denser arrangement than previously observed above, an abundance of immature matrix distributed among polymer fragments, and clusters of more mature-looking hyaline cartilage approaching the classic histological appearance of a chondrocyte within a lipid-filled lacuna.

Figure 5 represents the histological analysis of the construct that was just described, after it was transferred from the spinner flask to the STLV. This construct was allowed to incubate in the presence of FCS for an additional 12 days (ie, total cultivation time of 17 days), in the solid-body rotation mode, before breaking up into fragments during handling at medium exchange. An abundance of friable immature matrix is observed among the polymer fragments. The matrix histomorphological appearance appears similar to that noted during the 34-day spinner flask experiment that was described earlier in this section.

Figure 6 represents an 8-µm section of a construct that was seeded with cryogenically preserved chondrocytes at 8.6 × 106 cells per scaffold and cultivated in a spinner flask before being removed for analysis. The uniformity of seeding was by far the best compared with all other runs. A spectrum of matrix generation is evident from this figure. A cluster of hyaline cartilage with round chondrocytes in lipid-filled lacunae is evident.

DYNAMIC SEEDING KINETICS

Figure 7 shows the dynamic seeding data recorded from several measured bulk concentrations of chondrocytes (Table 1) by means of a hemocytometer (accurate to within 105 cells) as a function of time during the first 10 hours of seeding. All concentrations were normalized with the initial concentration to allow comparison between the various runs on a comparable dimensionless scale. All 6 subsets of data are represented by a unique curve, and these data came from independent runs, except for the 2 represented by the open square and diamond symbols, which were recorded from the same run. The serum-free run used 2 scaffolds at 11.0 × 106 cells per scaffold, control used no scaffolds at 13.3 × 106 cells, 2 scaffolds received 8.1 × 106 cells per scaffold, 1 scaffold received 43.7 × 106 cells per scaffold, frozen chondrocytes used 1 scaffold at 8.6 × 106 cells per scaffold, and 3 scaffolds received 9.6 × 106 cells per scaffold. Overall, seeding completeness, as defined by the normalized concentration measured at the end of the 10-hour measurement period, ranged between 40% and 95%. The data pertaining to the serum-free run were associated with the upper end of this scale, while the data representing the runs with fewer than 2 scaffolds, which included the cryogenically preserved cell line, were associated with the lower end. Seeding kinetics followed an exponential decay, independent of the presence or absence of serum. The seeding kinetics of the cryogenically preserved chondrocytes was also exponential.

MODIFIED TETRAZOLIUM-BASED COLORIMETRIC ASSAY

This assay, which is based on measuring the absorbance shift of the supernatant subsequent to reaction with the living cell's mitochondrial dehydrogenase, showed cell viability in 1 scaffold seeded at 8.1 × 106 cells per scaffold after 16 days' incubation in the spinner flask, which was agitated at 50 rpm.

SCANNING ELECTRON MICROSCOPY

Figure 8 shows a scanning electron micrograph of a 5-day construct just after being dynamically seeded in the spinner flask at 6.4 × 106 cells per scaffold. Adherent chondrocytes appear in the process of filling the void spaces straddling the polymer fibers with de novo extracellular matrix material.

COMMENT

Optimal distribution of anchorage-dependent cells in the interstices of the synthetic cell delivery vehicle minimizes the time spent in suspension by these cells and maximizes uniform cellular attachment to the substratum. Dynamic seeding of chondrocytes in the turbulent flow field of magnetically stirred spinner flasks has been shown to allow the most efficient means of delivering cells to the polymer scaffolds.20 This has been attributed to mixing associated with turbulent flow, which promotes the formation of a statistically uniform distribution of cellular aggregates followed by their deposition in the void spaces of the scaffold primarily by means of gravitational settling and direct interception. Aggregate formation is an early biological phenomenon that, through community effect, may be instrumental in driving neomorphogenesis.21 Recently, it has been shown that dynamic seeding of bovine chondrocyte aggregates of 20 to 30 µm follows exponential temporal kinetics.20 This has been correlated with aggregate formation in turbulent flow, which also has been shown to take place exponentially. Our results with human septal chondrocytes agree with these findings (Figure 7). Dynamic seeding time profiles showed near-complete exponential seeding within the first 10 hours of cultivation. Comparison of seeding behavior in a control flask (ie, no scaffold) with a 2-scaffold flask suggests that the scaffolds, as opposed to cell death or gravitational settling of cell aggregates, served as the preferential cell sink.

The data recorded from the serum-free study showed the highest kinetic rate (Figure 7). Differences in seeding density did not explain these observations. The more efficient attachment of human chondrocytes compared with bovine chondrocytes in serum-free media has been noted in the literature.22 However, enhanced human chondrocyte death has also been observed under serum-free conditions. These findings prevent us from determining which of the 2 mechanisms has been responsible for the observed accelerated kinetics in serum-free media. Furthermore, it is not clear what impact lack of serum factors may have on the rate of aggregate formation. By contrast, because of the absence of serum albumin, a cellular attachment mediator, from the media, the likelihood of cell attachment to the walls of the vessel would render this cell sink nonsignificant. One more piece of information, which further complicates this issue, is that the chondrocytes were amplified in the presence of serum and only during the seeding phase were they deprived of it. It is entirely possible that, during the short-lived seeding phase, the chondrocytes may still have retained their serum-attributed physiological changes (ie, expression of cell attachment receptors) that were acquired during the amplification phase. Further study is needed to resolve this issue.

In contrast to the observed serum-free rates, the data representing the runs with fewer than 2 scaffolds, which included the cryogenically preserved cell line as well, were associated with the lowest kinetic rates. The seeding kinetics of the cryogenically preserved chondrocytes, however, were found to be exponential as well. No detectable difference was observed between the rates of decrease of measured bulk concentration when either 1 or no scaffold (ie, mixed cell suspension, control) was used. This observation was independent of the seeding density. By contrast, the 2- and 3-scaffold results were correlated with seeding density (Figure 7, the lower curves represented by the open triangle, diamond, and circle symbols), albeit through an inverse relationship. The lack of an observed direct relationship with scaffold number, as has been previously reported,20 suggests that the hemocytometer may not be sensitive enough to detect these changes at our level of cell density, which measured many orders of magnitude below those used by these investigators. By virtue of using rapidly expandable animal cell sources, these investigators were able to achieve initial densities approximating 80 × 106 chondrocytes. A discernible difference was also evident in the kinetic rates of the 2- and 3-scaffold setups and those in which only 1 or no scaffold was mounted, such that the bulk concentrations decreased at a faster rate when more than 1 scaffold was present. Overall, the data distinguish between the seeding kinetics of human septal chondrocytes in serum-free vs serum-based media, the latter likely being more efficient than the former. In addition, the data are consistent with the law of conservation of mass, as reflected by the faster cell seeding observed when more than 1 scaffold was used to trap the chondrocytes. Although the cryogenically preserved chondrocytes followed the lower extreme of cell seeding kinetics, further studies are needed to determine if this has resulted from the compromised nature of these cells, as has been previously reported.23 The fact that histological sections of these chondrocytes showed adequate uniform seeding along with proper matrix regeneration lends less support to compromised cell physiology.

Constructs that were seeded with 2.3 × 106 cells per scaffold and were cultivated in a spinner flask in the presence of larger FCS volume (250 mL) for 34 days appeared friable, soft textured, collapsible under gravity, neutrally buoyant in media, easily breakable on handling, and grayish white in gross appearance, but did not fall apart during the entire run, as previously reported in the literature.20 Because of the observed compromised sensitivity of hemocytometry at increased media volumes, all subsequent runs were carried out at reduced volumes. However, chondrocyte-polymer constructs that were independently seeded at 8.1 × 106 cells per scaffold in a reduced medium volume (120 mL), under otherwise identical conditions, buckled on seeding and became nearly of pastelike consistency before dissolving into medium after 17 days' incubation. Because we used unreinforced thin PGA scaffolds, structural collapse under excessive cell load would offer an alternative explanation to early breakdown because of local acid buildup. In addition, excessive hydrodynamic stress caused by increased viscous dissipation as a result of using a smaller medium volume could have resulted in faster polymer breakdown and cell death, leading to collapse under otherwise identical conditions. Given identical inoculation cell density, a reduced medium volume would result in increased kinematic viscosity. This would then decrease the size of the smallest turbulent pockets (eddies), known as the Kolomogrov length scale.24 The smaller eddies, which are known to be more likely to damage cells, may have contributed to the observed accelerated structural collapse.

In contrast to constructs that were discussed above, those that were transferred into an STLV after 1 week or less of incubation time in a spinner flask appeared to have a smooth yellowish gray surface and rounded corners. One of these constructs, which was seeded at 9.6 × 106 cells per scaffold, continued to remain in a buckled configuration. This buckling occurred during the spinner flask phase of cultivation. By contrast, another construct that had been independently seeded at 43.7 × 106 cells per scaffold appeared more flexible as it regained its original flattened-out configuration during the STLV phase of cultivation. Because of the presence of attenuated shear stress in the flow fields of the STLV and elimination of the gravitational vectors,25 it is likely that the detrimental hydrodynamic forces that dominate in the turbulent mixing environment of the spinner flasks were instrumental in early cell death and structural collapse. These forces in particular may become cumulatively more damaging at longer culture times, arguing in favor of quiescent growth in simulated microgravity after dynamic seeding.

Histological observations showed uniform seeding and presence of basophilic matrix, especially in the immediate neighborhood of the polymer scaffold fibers (Figure 2). Pyknotic nuclei, distributed throughout the chondrocyte-polymer constructs at later time points, subsequently became replaced by matrix as evidenced by the increasing intracellular distance. This event is a natural outcome of neochondrogenesis. Islands of hyaline cartilage with rounded chondrocyte morphological characteristics embedded in lipid-filled lacunae were detected as early as 72 hours of cultivation time in the spinner flasks. These random patches of mature cartilage were mostly detectable on the peripheral zones. The gradient of matrix formation from the peripheral to the more central zones is likely caused by selective seeding of the former.26 Alternatively, the presence of randomly distributed clusters of matrix in addition to their peripheral presence may be caused by appositional as well as interstitial growth characteristics of hyaline cartilage. The constructs that were grown at lower than 2.3 × 106 cells per scaffold showed rough-textured matrix histomorphological characteristics (Figure 3) that were less basophilic than expected of fully matured ground substance. It is likely that low chondrocyte density compromised proper cell-cell contact, with consequent immature matrix production. In addition, these constructs were incubated in the spinner flasks for 34 days. The long-term exposure to turbulent stress may have diverted the cell metabolic energy toward manufacturing of shear-resistant matrix components, compromising mature ground substance formation. Specific immunoperoxidase matrix antibody staining may shed more light on this issue. Lack of mature matrix production was also evident from constructs that were incubated in the STLV for 12 days after being seeded with 8.1 × 106 cells per scaffold in the spinner flasks for 5 to 7 days. Although friable-appearing and histomorphologically less mature matrix was found to be more abundant (Figure 5), this was more likely because of the use of higher initial cell density than the 3-dimensional cell culture environment. Moreover, because of the absence of relative velocity between the medium and the construct during solid-body rotation, this mode of matrix regeneration may be inferior to tissue growth under true simulated microgravity. Further studies are needed to resolve these issues.

The described method has direct clinical applicability in facial, middle ear (conductive hearing transducer), and airway reconstruction. Perhaps its strongest role will be laryngeal and tracheal reconstruction, such as in repair of subglottic stenosis and tracheal resection for cancer. For this we envision taking a small piece of septal cartilage from the patient and then growing the cartilage in a template representing the deficient region. The implant will have more structural support, more accurate contour because of template formation, and less morbidity than the traditional rib graft. This system also has applicability in nasal reconstruction for dorsal nasal collapse (saddle nose) and nasal valve collapse. Also, harvested and expanded cartilage can be used to repair facial defects after trauma (eg, hemifacial asymmetry). Furthermore, auricular reconstruction (eg, microtia and traumatic loss) may be revolutionized.

In summary, human septal chondrocytes may be obtained in a sterile fashion from patients undergoing septoplasty. Once combined with optimally designed PGA scaffolds at the appropriate cell density in the bioreactor cascades that allow efficient seeding and quiescent tissue growth, these chondrocytes become a valuable source of reproducible ex vivo cartilage regeneration in the laboratory. Although we have been able to show evidence of hyaline cartilage formation with this method, many issues remain that require resolution before we leap into excluding serum from the cell culture medium and replacing the important factors. In particular, we will be conducting experiments to address the following issues: (1) the benefit of true simulated microgravity, (2) potential use of cryogenically preserved chondrocytes as the cell source, and (3) effect of serum-free medium on dynamic seeding and tissue formatting before implantation.

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Article Information

Accepted for publication September 30, 1999.

Presented at the 1999 Annual Meeting of the American Academy of Otolaryngology–Head and Neck Surgery, New Orleans, La, September 27, 1999.

We acknowledge Jon Kosek, MD, professor of pathology, for histological analysis of the cartilage constructs. Thanks are also due to Nicole Baumgardth, PhD, postdoctoral fellow, Department of Genetics, for technical assistance with the modified tetrazolium-based colorimetric assay.

Reprints: R. James Koch, MD, MS, Division of Otolaryngology–Head and Neck Surgery, Edwards Building, Room 135, Stanford University Medical Center, Stanford, CA 94305-5328 (e-mail: rjk@stanford.edu).

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