Propagation of Human Nasal Chondrocytes in

Propagation of Human Nasal Chondrocytes in Microcarrier Spinner Culture Delivered by Ingenta to Alan H. Shikani, M.D.,* David J. Fink,IP: Ph.D.,§ Afshin Sohrabi, M.S.,# Phong Phan, B.S.,# 65.111.73.50 Anna Polotsky, M.D.,# David S. Hungerford, M.D.,# and Carmelita G. Frondoza, Ph.D.#

ABSTRACT Objective: The aim of this study was to test the effectiveness of nasal septal chondrocytes, propagated in microcarrier spinner culture, as an alternative tissue source of chondrocytic cells for cartilage grafts for head and neck surgery and for articular cartilage repair. Methods: We harvested chondrocytes from 159 patients, ranging in age from 15 to 80 years and undergoing repair of a deviated nasal septum, and propagated the cells in a microcarrier spinner culture system. The nasal chondrocytes proliferated and produced extracellular matrix components similar to that produced by articular chondrocytes. Results: In microcarrier spinner culture on collagen beads, chondrocyte numbers increased up to 14-fold in 2 weeks. After a month, the microcarriers seeded with nasal chondrocytes

From the Departments of *Otolaryngology and #Orthopaedic Surgery, Johns Hopkins University, Baltimore, Maryland, and §Chondros, Inc., Baltimore, Maryland Presented at the annual meeting of the American Rhinologic Society (ARS), San Diego, CA, 2002 Supported in part by The Johns Hopkins University Orthopaedic Rheumatology Gift Fund, The Good Samaritan Hospital, and Chondros, Inc. A.H. Shikani, M.D., D.J. Fink, Ph.D., A. Sohrabi, M.S., D.S. Hungerford, M.D., and C.G. Frondoza, Ph.D., are stockholders in Chondros, Inc. Address correspondence and reprint requests to Carmelita G. Frondoza, Ph.D., Department of Orthopedic Surgery at Good Samaritan Hospital, Professional Office Building G-1, 5601 Loch Raven Boulevard, Baltimore, MD 21239 Copyright © 2004, OceanSide Publications, Inc., U.S.A.

American Journal of Rhinology

began to aggregate, producing a dense cartilage-like material. The newly synthesized extracellular matrix was rich in high molecular weight proteoglycans, and the chondrocytes expressed type II collagen and aggrecan but not type I collagen. Conclusion: These studies support the feasibility of engineering cartilage tissue using chondrocytes harvested from the nasal septum. Injectable and solid formulations based on this technology are being evaluated for applications in craniomaxillofacial reconstructive surgery and for plastic and orthopedic surgery practices. (American Journal of Rhinology 18, 105–112, 2004)

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he first reported use of a cartilage autograft in humans was by Ko¨nig in 1896, and since then, sculpted cartilage grafts have been used widely as facial implants. Over the last 20 years, the use of tissue-engineered cartilage involving the combinations of chondrocytes and different biomaterials has been studied actively to reconstruct diseased or damaged tissues in the fields of craniomaxillofacial plastic reconstructive surgery, head and neck surgery, and orthopedic surgery. Limited tissue availability and donor site morbidity are drawbacks when using the patient’s own cartilage. Banked homologous cartilage has been used but it has a faster resorption rate than autologous cartilage and the resorption rate varies depending on the method of preservation and the site of implantation.1 Allogeneic chondrocyte suspensions have been used, but there are concerns with immune rejection and possible disease transmission. Autologous tissue-engineered cartilage was first used in 1989, transplanting chondrocytes alone or in combination with biocompatible material, and since then, extensive research has been done using chondrocytes with a variety of different polymers. Recently, marrow-derived stem cells that are capable of differentiating into chondrocytes have been investigated.2 ,3 In clinical practice, the closer the properties of the tissueengineered cartilage to the native cartilage, the higher the

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probability of clinical success. For example, in orthopedic formulation of autologous human injectable tissue-engisurgery a widely used source of cells for joint cartilage neered cartilage derived from donor sites that are easily repair is articular cartilage from a healthy joint. Articular accessible without significant morbidity such as the nasal chondrocytes synthesize the requisite matrix molecules, septum. We have described a microcarrier bioreactor spintype II collagen, and aggrecan, which are essential for ner culture system1 2 that has been used to propagate articsuccessful resurfacing of an articular defect. However, the ular chondrocytes from donors undergoing joint replaceclinical limitation is the rarity and limited distribution of ment and nasal chondrocytes from patients undergoing healthy articular chondrocytes in the body, and harvesting repair of a deviated nasal septum. This system has been cartilage from healthy joints eventually may lead to degrashown previously in our laboratory to maintain nasal cell dation of the tissues surrounding the harvest site.4 Moreviability and chondrocytic phenotype while enhancing proover, an open arthrotomy is required for cartilage harvestliferation.1 2 Moreover, cells previously grown in monolayer ing, which is invasive and expensive, hence the interest in culture and shown to “dedifferentiate” to a fibroblastic other sources of cartilage. Delivered by Ingenta phenotype to were shown to regain their chondrogenic phenoIn facial plastic reconstructive surgery, the nasal IP:septum, 65.111.73.50 type when placed in spinner culture.1 2 In the spinner culture the auricle, and the ribs have all proven to be successful system, nasal chondrocytes proliferated and produced exdonor sources for sculptable solid cartilage grafts, the nasal tracellular matrix (ECM) components similar to that proseptum being the most accessible with the least amount of duced by articular chondrocytes, including high levels of morbidity. Cells harvested from the nasal septum of a patype II collagen, aggrecan, and b1-integrins,1 3 with low type tient have been shown to grow successfully in a threeI collagen levels, which makes them suitable both for ordimensional polymer scaffold made of polyglycolic acid.5 ,6 thopedic and for head and neck applications. Based on this Successful seeding of three-dimensional scaffolds has also technology, both injectable and solid cartilage formulations been demonstrated using articular chondrocytes. Scaffolds are under development. are important when resurfacing articular joints because they can be made to fill the void volume of the cartilage defect MATERIALS AND METHODS precisely and fix in the defect to withstand the forces of Preparation of Nasal Chondrocytes joint motion. On the other hand, in head and neck applications, precise fixation is not as important but extrusion and iscarded nasal cartilage tissue was obtained from 159 polymer biocompatibility are major considerations. The patients undergoing nasal septum reconstruction. The most commonly used polymers, polylactic acid and polytissue was soaked in Betadine for 10 minutes and then glycolic acid and their copolymers have had good chondrominced into pieces ;1 mm in size.3 Chondrocytes were cyte biocompatibility but have been shown to cause a forisolated by digestion in 0.1% (w/v) collagenase (Boehringer eign body giant cell reaction, which may limit chondrogenMannheim, Mannheim, Germany) for 24 hours at 37°C and esis.1 ,7 Other problems have included nonuniform 5% CO2 with constant stirring. The enzymic digestion was chondrocyte seeding of the scaffold mesh because of inconstopped by washing the digest twice with Hank’s balanced salt sistency in the polymer porosity, limitation in the size of the solution. Individual chondrocytes were separated from undiscaffold because of nutrient transport during culture, and gested cartilage and cartilage debris using the Cellector tissue dedifferentiation of the implanted chondrocytes to a fibrosieve (VWR International West Chester, PA). blastic phenotype, which led to undesirable fibrous tissue Evaluation of Cell Viability and Number formation and even bone.1 ,8 In an attempt to avoid the foregoing problems associated ells in trypan blue dye were enumerated using a hewith cell-seeded preformed solid polymer scaffolds and the macytometer, and unstained cells that did not take up need for the open surgical procedure required for precise the dye were considered viable. Morphology was studied positioning of the implant, research has been performed on using phase-contrast microscopy. injectable polymer-chondrocyte formulations, which can be Propagation of Nasal Chondrocytes on Microcarriers introduced using minimally invasive techniques such as injection with a large bore needle under endoscopic guidhis technique has been described previously in detail ance either into the joint space or subcutaneously for craniofor the propagation of human articular chondrocytes; facial reconstruction and recontouring. There is a paucity of the procedure used to culture human nasal chondrocytes is research on injectable tissue-engineered cartilage. Pig articessentially the same.1 2 Briefly, cells were propagated as ular cartilage cells have been suspended is a fibrin glue monolayer cultures in enriched Dulbecco minimal essential polymer carrier and survived when injected subcutaneously medium (Sigma-Aldrich, St. Louis, MO) supplemented in nude mice.9 Pig chondrocytes also have been delivered in with 20% fetal calf serum (Gemini Bioproducts, Woodland, a hydrogel carriers including calcium alginate and pluronCA) until confluence. In cases in which very small tissue ics.1 0 Molded chondrocyte/alginate constructs in the shape samples were available, cells were replated until at least 4 of facial implants have been described recently.1 1 million cells were available. The cells were harvested by The purpose of this research is to create an improved trypsinization, counted, and assayed for viability. Chondro-

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Figure 2. Photomicrograph of nasal cartilage. Section of tissue Delivered by Ingenta to was stained with hematoxylin and eosin and photographed at low IP: 65.111.73.50 and high magnifications. Figure 1. Schematic diagram of chondrocyte isolation and propagation in Cellagen microcarrier spinner culture.

cytes were seeded subsequently onto collagen microcarrier beads (Cellagen, 100–400 mm in diameter, derived from bovine corium; ICN, Cleveland, OH) at a density of 4 3 103 chondrocytes/cm2 in a siliconized spinner flask. During the first 4 hours, the mixture was stirred intermittently for 2 minutes every 30 minutes at 25–30 rpm. The cell-microcarrier suspension was stirred subsequently at 45 rpm for another 4 hours. The speed was increased gradually to 60 rpm and then maintained at 60 rpm for the duration of the experiment. The final volume of the suspension culture was 30 mL per 1.0 3 106 chondrocytes. To replenish the spinner cultures, the microcarriers were sedimented for 5 minutes and ;50% of the spent medium was replaced every 3 days. Spinner cultures were incubated at 37°C, 5% CO2. At 90% confluence, the cell-coated microcarriers were removed aseptically and retrieved using 0.1% collagenase, counted, and assayed for viability. Approximately 3.0 3 106 nasal chondrocytes were frozen at 270°C and used for semiquantitative reverse transcriptase–polymerase chain reaction (RT-PCR) analysis to verify the presence of specific chondrocytic phenotype markers. The remaining chondrocytes were reseeded onto Cellagen microcarriers. These third-passage nasal chondrocytes were allowed to reach 90% confluence and then used for the phenotypic analysis using immunoperoxidase. Figure 1 outlines the tissue culture methods used in this experiment. RNA Extraction and Analysis by RT-PCR otal RNA was isolated by the TRIzol (Life Technologies, Rockville, MD) reagent method. A total complementary DNA (cDNA) library was synthesized using the advantage RT-PCR kit (Clontech Laboratories, Palo Alto, CA) and Oligo (dT18) primer.1 4 The resulting RT product was expanded using the SuperTaq Plus (Ambion, Austin, TX) PCR kit and specific primers for types I and II collagen, aggrecan, and the housekeeping gene ribosomal RNA S14 subunit. Two microliters of cDNA template were used in

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each PCR reaction. The PCR products were analyzed by agarose gel electrophoresis, and densitometry was performed using the UN-SCAN-IT Gel Automated Digitizing System (Silk Scientific Corp. Orem, UT).1 5 – 1 7 Histological and Cytological Analysis artilage pieces or cultured chondrocyte-microcarrier aggregates were fixed in 90% methanol, paraffin embedded, and cut into 7-mm sections. The prepared sections were deparaffinized and stained with hematoxylin and eosin. Figure 2 shows a photomicrograph of histology for a typical nasal cartilage section. Chondrocytes were allowed to reach 90% confluence and then were fixed directly on their microcarriers for immunoperoxidase analysis of phenotype. Chondrocyte-seeded microcarriers were sedimented in conical tubes and then aliquoted onto microscope slides. Viability of chondrocytes was determined using trypan blue vital dye. Chondrocytes were transferred to microscope slides, fixed with 2% paraformaldehyde, and air-dried. Next, chondrocytes on microcarriers were immunostained with monospecific antibodies against types I and II collagen (Fisher Scientific, Pittsburgh, PA), and keratan sulfate (ICN Biomedicals, Inc., Aurora, OH). Staining was visualized using the immunoperoxidase technique with diaminobenzidine as substrate, which yielded a brownish color (Vector Laboratories Immunoassay Kit Brochure, Burlingame, CA). Cell preparations were counterstained with 0.5% toluidine blue. Human osteoblasts at passage one served as the positive cell control for type I collagen. Negative cell controls for types I and II collagen consisted of human lymphocytes. The specificity of the immunoperoxidase staining was verified by omitting the primary antibody. There was no staining when the primary antibody was omitted.

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RESULTS Chondrocyte Yield ince 1998, we have investigated 159 samples of nasal septal tissue in our laboratory in studies involving cell isolation, cell culture in the spinner culture system, and

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TABLE I Summary of Cell Yields from all Samples (n 5 70) and the Most Recent 13 of Samples (n 5 26) All Data

Male donors (n 5 44) Female donors (n 5 26) All donors (n 5 70)

Most Recent 13 of Samples

Mean Cell Yield (Million Cells/Gram)

Coefficient of Variation (%)

Mean Cell Yield (Million Cells/Gram)

Coefficient of Variation (%)

1.11 1.16 1.13

94.6 91.5 92.7

1.33 1.28 1.30

55.4 91.2 71.9

Coefficient of variation 5 SD/Mean (%).

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Figure 3. Cell yield from nasal septal cartilage for the last 26 tissues processed (14 male and 12 female donors).

Figure 4. Correlation of cell yield per gram with donor age for the last 26 nasal septal samples evaluated.

characterization of the cultured chondrocytes and resulting neotissues. Initially, problems with infections were encountered, and 35 samples were lost due to contamination or lack of growth. The rate of infection was significantly reduced by soaking the harvested cartilage in Betadine for ;10 minutes just after harvesting and then soaking for another 1 minute in Betadine just before isolating the chondrocytes. Contamination was reduced further by adding antibiotics (gentamicin) to the culture medium. Since 2000, isolation and cell culture protocols have been standardized and a total of 70 samples (44 males and 26 female donors ranging in age from 15 to 80 years) have been successfully cultured under these conditions. Cell yields from all of these 70 tissue specimens and for the latest one-third (26 total) of the male and female specimens are summarized in Table I. The correlation of cell yield with the weight of the tissue processed for the latest 26 samples is shown in Fig. 3. No correlation was observed when the complete data set was analyzed (data not shown; n 5 70; R2 5 0.018). The correlation of cell yield per gram of tissue with the age of the donor for the latest 26 cases is shown in Fig. 4.

Chondrocyte Growth Characteristics in Microcarrier Spinner Culture uman nasal chondrocytes obtained from septal cartilage (Fig. 2) attached and proliferated on microcarrier spinner culture. The doubling time for cell proliferation was ;4 days. The mean viability was .95% at each cell harvesting. No decrease in proliferative ability was observed, as determined by cell counting, or in cell viability, as determined through the vital dye exclusion assay. Table II summarizes the spinner culture cell yield, the number of cells obtained after 2 weeks of spinner culture, for the experiments performed to date. An average 5.8-fold increase in cell number was observed at 2 weeks and no correlation of cell yield with donor age or sex was observed in this limited series. Since the amount of tissue obtained from septal cartilage is fairly small, the retrieved chondrocytes were propagated normally first in monolayer culture from two to five passages (#2 months) depending on the mass of the original cartilage sample. As we have previously reported, articular and septal chondrocytes dedifferentiate in monolayer culture and then redifferentiate when

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TABLE II Examples of Cell Yields for Selected Cultures Donor Age Initial Cells Cell Yield After Cell Number (Years) Seeded 2 Weeks Increase (Millions) (Millions) (Fold) 38 38 44 45 49 58

4.0 3.5 4.0 4.5 4.0 4.0

25 18 20 25 30 22

6.3 5.1 5.0 5.5 7.5 5.5 Delivered

Figure 6.to Photomicrograph of aggregated microcarriers with by Ingenta adherent nasal chondrocytes after 15 days in spinner culture. The IP: 65.111.73.50

cells, mostly appear spherical, with eccentric nuclei, and are surrounded by matrix-like material. Cellagen microcarrier beads range in diameter from 100 to 400 mm.

Figure 5. Phase contrast photomicrograph taken at day 7. Nasal chondrocytes are attached to a Cellagen microcarrier.

propagated in spinner culture.1 2 , 1 3 On obtaining a sufficient number of cells from monolayer culture (;4 3 106 cells), microcarriers were seeded and the number of cells was determined after 2 weeks of culture. The examples in Table II include cases of reseeded chondrocytes after two to five passages in the monolayer culture. Nasal chondrocytes seeded in the spinner culture attached readily to collagen microcarrier beads during the initial intermittent stirring phase of the seeding process. By day 7, most microcarriers exhibited adherent cells on their surface (Fig. 4, indicating $80% plating efficiency). The chondrocytes proliferated on the surface of the Cellagen beads, attaining 90% confluence in ;8 days. At ;7 days, some chondrocyte-seeded Cellagen beads began adhering to other cell-coated beads. The multi-bead aggregates ranged from two cell-seeded beads to more than a dozen beads. The larger aggregates were clearly visible with the naked eye on macroscopic evaluation. On day 15 the chondrocyte-coated beads revealed the presence of mostly spherical-looking chondrocytes (Fig. 5). These cells had abundant cytoplasms and eccentric nuclei. There was deposition of ECM-like material around the beads and in the regions of bead-to-bead adhesion.


Evaluation of Nasal Chondrocyte Phenotype RT-PCR Analysis of Messenger RNA Levels for Collagens and Aggrecan. RT-PCR analysis of a typical recent culture of nasoseptal chondrocytes is shown in Fig. 6. The messenger RNA (mRNA) message for type II collagen was the most abundant of the phenotypic mRNA markers assayed. On electrophoretic analysis, the intensity of this ;350-bp transcription product was slightly greater than that of the housekeeping gene S14 (;130 bp). In contrast, the intensity of the type I collagen transcription product (;300 bp) was ,10% that of the housekeeping gene. The aggrecan-specific transcript had 13% of the intensity of the housekeeping gene. At the time the cells were harvested from the microcarrier, mRNA levels for type II collagen were much higher than those for type I collagen (13:1 ratio). The mRNA levels for the protein core of aggrecan were ;13% of the S14 housekeeping gene levels. RT-PCR has been performed successfully on 14 samples to date. Phenotypic profiles showed detectable levels of type II collagen and aggrecan and no or very low detectable levels of type I collagen. Cytological Analysis. The nasal chondrocyte-microcarrier aggregates stained intensely for both type II collagen and keratan sulfate (one of the sulfate-containing carbohydrate moieties of normal aggrecan). Staining was uniformly dark for both type II collagen and keratan sulfate over the surface of the cell-covered beads. However, the most intense staining was observed in the regions of dense ECM connecting one cell-covered bead to another. In contrast, staining for type I collagen was negligible, and omitting the primary antibody resulted in only background staining. Histological staining of cell-seeded microcarriers further illustrated that chondrocytes proliferated on the surface of the beads and produced ECM-like material. Figure 7 shows the hematoxylin and eosin staining of microcarriers seeded with nasal chondrocytes. Figure 8, A shows a photomicrograph of two batches of cells following cultures for 31 and

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Figure 7. Phenotypic expression of human nasal chondrocytes analyzed by RT-PCR. Chondrocytes harvested from the microcarDelivered rier spinner culture at 90% confluence were frozen at 270°C, and by Ingenta IP: 65.111.73.50 RNA extraction was performed for RT-PCR analysis. Row 1, expression of mRNA for the S14 housekeeping gene. Electrophoretic profiles were visualized on an agarose gel. Row 2, expression of mRNA for type II collagen. Note the high intensity of this type II collagen band. Row 3, expression of mRNA for type I collagen is negligible. Row 4, expression mRNA for aggrecan.

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28 days, respectively. Figure 8, B is a photomicrograph of a different line of cells and ECM surrounding several beads following 28 days of culture. Figure 8, C shows a highermagnification photomicrograph of beads surrounded by cells and ECM following the culture for 11 days. DISCUSSION e have evaluated chondrocytes derived from over 159 patients undergoing nasal septal reconstruction in a microcarrier spinner culture system and found that nasal chondrocyte proliferation was facilitated, with doubling times of ;4 days, and the chondrocytic phenotype was maintained. Cell yields from these tissues have improved as we have gained experience with processing nasal septal tissues. Typical cell yields for these specimens is 1.3 million cells per gram of nasal septal tissue. No correlation with donor age or sex and the cell yield have been observed to date. No correlation of cell yields in spinner culture (typically five- to sevenfold increases in cell number in 15 days) have been observed with donor age or sex in the limited series of spinner cultures performed to date. The dynamic mechanical nature of the culture system seems to help promote proliferation and the chondrocytic phenotype. The cells are grown on the surface of beads (100–400 mm in diameter) that are maintained in suspension by a stirrer, so that fluid shear is continuously affecting the cells and allows the cell-covered beads to collide with each other and with the siliconized walls of the culture flask. Nasoseptal chondrocytes, similar to articular chondrocytes, benefit from a mechanically active tissue culture environment.1 8 ,1 9 Thus, the ability to respond to a mechanical stimulus may be a characteristic of chondrocytes, irrespective of tissue source. Culture conditions for mammalian cells, including human cells, generally are thought to require gentle methods for suspending the cells because these

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Figure 8. Photomicrograph of nasal chondrocytes on Cellagen beads stained by hematoxylin and eosin at increasing magnification. (A) Chondrocyte-microcarrier aggregates cultured for 31 days (marker bar 5 100 mm). (B) Chondrocyte-microcarrier aggregates cultured for 31 days (marker bar 5 100 mm). (C) Chondrocyte-microcarrier aggregates cultured for 31 days (marker bar 5 100 mm).

cells are unusually sensitive to turbulent fluid forces. However, for chondrocyte culture, the unexpected observations in spinner culture indicate that these cells respond to these highly mixed conditions by secreting additional cartilagelike matrix molecules and stabilizing their microenvironments. In most tissue-engineering systems in which cells are cultured on porous scaffold devices, the thickness of the construct is limited to 2–3 mm in the minor dimension


because nutrient and metabolite diffusion become rate-limmillion cells per gram of tissue), expand and multiply the iting in thicker constructs. This limitation has caused many cells in vitro in microcarrier spinner culture, maintain the researchers to investigate mechanical methods or novel bioproper chondrocytic phenotype, and, thereby, create de reactor designs to improve nutrient supply to tissue-enginovo cartilage for implantation. Reports from this laboraneered cartilage.2 0 ,2 1 An advantage of the microcarrier cultory have described previously related experiments comparture conditions is that during most of the culture period, the ing static (monolayer) and dynamic (spinner) cultures of cell-microcarrier aggregates are suspended independently, articular and nasal chondrocytes.1 2 ,1 3 In these studies, spinand therefore nutrient supply to the cells is maximized. ner cultures enhanced cell proliferation and retention of Under these conditions, matrix secretion is increased and phenotype from both sources of cells compared with monoculture time is minimized. The ECM formed by the chonlayer cultures. Currently, we are working to standardize the drocytes in the microcarrier spinner culture was rich in type method of processing nasal cartilage tissue and to assess the II collagen, whereas type I collagen was virtually undetectoptimal amount of cartilage to biopsy. Based on this expeable. These observations suggest the involvementDelivered of a tran- by Ingenta to believe that healthy chondrocytes isolated from rience, we scriptional mechanism geared toward the maintenance IP: 65.111.73.50 of nasal septum can provide a reliable source of cells for use in the chondrocytic phenotype. Askew et al.2 2 reported that the successful tissue-engineered cartilage implants. phenotypic shift undergone by chicken embryo chondrocytes is mediated at the transcriptional level. Additionally, REFERENCES Miosge et al.2 3 found a correlation between the levels of 1. Britt JC, and Park SS. Autogenous tissue-engineered cartilage. types I and II collagen and the phenotype of chondrocytes Arch Otolaryng. Head Neck Surg 124:671–677, 1998. using in situ mRNA hybridization technique on fibrocarti2. Wakatani S, Goto T, Pineda SJ, et al. Mesenchymal cell-based laginous tissue. The ECM also was shown by immunocyrepair of large full-thickness defects of articular cartilage and tochemical methods to contain sulfated proteoglycans. Inunderlying bone. J Bone Joint Surg 76:579, 1994. corporation of sulfate-containing sugar groups to the protein 3. Grande DA, Lucas PA, Manji R, et al. Repair of articular cartilage defects using mesenchymal stem cells. Tissue Eng backbone endows a hydrophilic nature to aggrecan and 4:345–353, 1995. gives cartilage its resistance to compression, which facili4. Grande DA, Breitbart AS, Mason J, et al. Cartilage tissue engitates weight bearing. neering: Current limitations and solutions. Clin Orthop 367S: Another characteristic of the microcarrier culture is the 176S–185S, 1999. constrained environment for propagating chondrocytes. 5. Naumann A, Rotter N, Bujia J, et al. Tissue engineering of This would resemble the state of chondrocytes in cartilage autologous cartilage transplants for rhinology. 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ORL J Otorhinolaryngol Relat substrate material (i.e., highly cross-linked type I collagen) Spec 55:347–351, 1993. also may contribute to the maintenance of the chondrocytic 9. Silverman RP, Passaretti D, Huang W, et al. Injectable tissuephenotype. engineered cartilage using fibrin glue polymer. Plast Reconstr If nasal chondrocytes are cultured in microcarrier spinner Surg 103:1809 –1818, 1999. 10. Cao Y, Rodriguez A, Vacanti M, et al. Comparative study of the culture for 10–14 days, an injectable cartilage formulation is use of poly(glycolic acid), calcium alginate, and pluronics in the formed, composed of the chondrocyte-microcarrier aggreengineering of autologous porcine cartilage. J Biomater Sci gates cultured. If the chondrocytes are allowed to spin in Polym Ed 9:475–487, 1998. microcarrier spinner culture beyond that time, more ECM is 11. Chang SCN, Rowley JA, Tobias G, et al. 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