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Expansion of human nasal chondrocytes on macroporous microcarriers enhances redifferentiation
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Biomaterials 24 (2003) 5153–5161
Expansion of human nasal chondrocytes on macroporous microcarriers enhances redifferentiation J. Maldaa,b,*, E. Kreijvelda, J.S. Temenoffc, C.A. van Blitterswijka,b, J. Rieslea b
a IsoTis N.V., P.O. Box 98, 3720 AB Bilthoven, The Netherlands Institute for BioMedical Technology (BMTI), University of Twente, P.O. Box 217, 7500 AE Enschede, The Netherlands c Department of Bioengineering, Rice University, Houston, TX 77251-1892, USA
Received 9 December 2002; accepted 23 May 2003
Abstract Articular cartilage has a limited capacity for self-repair. To overcome this problem, it is expected that functional cartilage replacements can be created from expanded chondrocytes seeded in biodegradable scaffolds. Expansion of chondrocytes in twodimensional culture systems often results in dedifferentiation. This investigation focuses on the post-expansion phenotype of human nasal chondrocytes expanded on macroporous gelatin CultiSpher G microcarriers. Redifferentiation was evaluated in vitro via pellet cultures in three different culture media. Furthermore, the chondrogenic potential of expanded cells seeded in polyethylene glycol terephthalate/ polybuthylene terephthalate (PEGT/PBT) scaffolds, cultured for 14 days in vitro, and subsequently implanted subcutaneously in nude mice, was assessed. Chondrocytes remained viable during microcarrier culture and yielded doubling times (1.0770.14 days) comparable to T-flask expansion (1.2070.36 days). Safranin-O staining from pellet culture in different media demonstrated that production of GAG per cell was enhanced by microcarrier expansion. Chondrocyte–polymer constructs with cells expanded on microcarriers contained significantly more proteoglycans after subcutaneous implantation (288.5729.2 mg) than those with T-flask-expanded cells (164.0728.7 mg). Total collagen content was similar between the two groups. This study suggests that macroporous gelatin microcarriers are effective matrices for nasal chondrocyte expansion, while maintaining the ability of chondrocyte differentiation. Although the exact mechanism by which chondrocyte redifferentiation is induced through microcarrier expansion has not yet been elucidated, this technique shows promise for cartilage tissue engineering approaches. r 2003 Elsevier Ltd. All rights reserved. Keywords: Nasal chondrocytes; Redifferentiation; Polyethylene glycol terephthalate/polybuthylene terephthalate (PEGT/PBT); Macroporous microcarriers; Insulin; Tissue engineering
1. Introduction Because of the limited self-renewal capability of articular cartilage, an estimated one million people per year require treatment of cartilage defects [1]. Once damaged, these defects do not heal spontaneously. Therefore, tissue engineering approaches offer new possibilities for functional and structural restoration of the damaged or lost tissue. In the development of a tissue engineered implant for the treatment of articular cartilage defects several requirements should be considered. In order to functionally and mechanically restore the defect, the implant *Corresponding author. 0142-9612/$ - see front matter r 2003 Elsevier Ltd. All rights reserved. doi:10.1016/S0142-9612(03)00428-9
should include reparative cells capable of synthesising hyaline cartilage-specific extra-cellular matrix (ECM), supported by a biodegradable scaffold that matches the mechanical properties of the surrounding tissue. While a variety of cell sources may be used in the construct, an autologous patient biopsy can be preferable as this decreases the risk of immunogenic response and disease transfer. Articular cartilage from little loadbearing regions could be used, but, due to donor site morbidity, the amount of tissue is limited. Therefore, the use of alternative autologous cell sources, such as human septum cartilage, has been evaluated [2,3]. Harvest of cartilage from the nasal septum requires less invasive surgery then harvest from articular sources and the resulting morbidity is minimal [2]. Furthermore,
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Fig. 1. Experimental design. Isolated human nasal chondrocytes were expanded for 12 days in T-flasks or on CultiSpher G microcarriers. To evaluate the post-expansion chondrocytic phenotype, the resulting cells were seeded either in pellets or on PEGT/PBT scaffolds and cultured for 7 and 14 days, respectively. Furthermore, PEGT/PBT-cartilage constructs were implanted for an additional 14 days subcutaneously (s.c.) in nude mice.
several investigators have reported increased cartilage formation by nasal septum chondrocytes in comparison to articular chondrocytes and indicated the potential of nasal cartilage as a cell source for cartilage tissue engineering strategies [2,3]. To reduce the size of the required biopsy, prior to seeding on scaffolds, cells must be expanded to obtain a sufficient cell number. During this process, the chondrocytes loose their spherical shape and acquire a fibroblast-like appearance [4,5]. Accordingly, expression of hyaline cartilage markers, aggrecan and collagen type II, decreases, while expression of non-hyaline cartilage specific collagen type I increases [5]. In an effort to overcome this problem, the culture of chondrocytes on microcarriers, has been investigated as a means to maintain a chondrocytic phenotype, resulting in specific expression of collagen type II, aggrecan and beta-1 integrin [6–9]. Additionally, we recently demonstrated that expansion of immature bovine chondrocytes on non-porous Cytodex 1 microcarriers increased neo-cartilage formation in subsequent pellet cultures, as compared to pellets of chondrocytes expanded in T-flasks (Malda et al., in press). Macroporous microcarriers, including gelatin-based carriers, offer the advantage that the surface area per bead can be further increased, thus allowing the attachment of more cells per carrier [10,11]. The use of gelatin-based macroporous microcarriers is also advantageous in that, since the beads are susceptible to proteolytic enzymes, such as trypsin, harvest of expanded cells is greatly simplified. Therefore, in this study, human septal chondrocytes were cultured on marcoporous CultiSpher G gelatin microspheres prior to phenotypic analysis. In addition to reparative cells, the scaffold plays a pivotal role in cartilage tissue engineering. It serves as a template to guide the growth and organization of the
developing tissue and gives stability to the neo-tissue [12]. Our method adopts a porous biodegradable segmented-block-copolymer scaffold. The biocompatibility of this material, which is composed of polyethylene glycol terephthalate (PEGT) and polybutylene terephthalate (PBT) segments, has been demonstrated in several studies [13–20]. By varying the ratio of the alternating soft (PEGT) and hard (PBT) segments the mechanical properties of this material can be tailored [21] to match the mechanical properties of cartilage [22]. In the present studies, the post-expansion phenotype of human nasal chondrocytes expanded on macroporous gelatin CultiSpher G microcarriers was investigated (Fig. 1). Neo-cartilage formation was evaluated in vitro via pellet culture [23] in three different media. Furthermore, the potential of expanded cells was assessed after seeding in PEGT/PBT copolymer scaffold culture for 14 days in vitro, and subsequent subcutaneous implantation in nude mice. Proteoglycan production in the cellpolymer constructs was evaluated histologically via safranin-O staining at various time points, while proteoglycan and total collagen production were quantified concurrently using biochemical assays.
2. Materials and methods 2.1. Cell isolation Nasal cartilage was obtained from patients (n ¼ 9; age range: 18–51 years, average: 36 years) undergoing nasal septum reconstruction. Cartilage was dissected in approximately 1-mm3 cubes. Chondrocytes were isolated by overnight digestion with 0.15% type II collagenase (Worthington Biochemical) at 37 C. After three washes in phosphate-buffered saline (PBS)
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(Gibco-BRL), cells were suspended in medium 1 (HEPES-buffered DMEM (Gibco-BRL) supplemented with 10% FCS (Sigma-Aldrich), 0.2 mm ascorbic acid 2phosphate (Gibco-BRL), 0.1 mm non-essential amino acids (Sigma-Aldrich), 0.4 mm proline (Sigma-Aldrich), 100 units/ml penicillin (Gibco-BRL), and 100 mg/ml streptomycin (Gibco-BRL) and either seeded in T-flasks or microcarrier culture. 2.2. Cell expansion 2.2.1. Microcarrier seeding and expansion Macroporous gelatin-based CultiSpher G microcarrier beads (Percell Biolytica), + 130–180 mm, were prepared according to the manufacturer’s instructions. In short, microcarriers were hydrated prior to use in PBS (Gibco-BRL) at 37 C and steam-sterilized (15 min, 121 C) in Sigmacote (Sigma-Aldrich) treated bottles. Microcarriers were then rinsed twice with PBS and once with 37 C medium 1 and transferred to a Sigmacote treated 250 ml spinner flask (working volume 100 ml, end concentration 1 mg CultiSpher G/ml). Microcarriers were cell-seeded (1.0 106 cells/flask) in 50 ml of medium 1 using an intermittent stirring regime (30 min at 0 rpm, 30 s at 20 rpm) for 24 h (n=2). After seeding, 50 ml of medium 1 was added and the stirring speed was increased to 50 rpm. All cultures were maintained at 37 C in a humidified 5% CO2 incubator. For daily cell counts, 0.5 ml samples were rinsed with PBS and 150 ml 0.25% Trypsin/1.0 mm EDTA (Gibco-BRL) was added to dissolve the microcarriers. After 15 min of incubation at 37 C, cells were counted using a particle count and size analyser (Coulter Corporation). For cell harvesting, microcarriers were allowed to settle for 10 min. The supernatant was removed and the culture was rinsed twice with PBS. Subsequently, 15 ml 0.25% Trypsin/1.0 mm EDTA was added to dissolve the microcarriers. After 15 min incubation at 37 C, 15 ml of culture medium was added and cells were counted using a particle count and size analyser (Coulter Corporation). Cell viability was measured using trypan blue (SigmaAldrich) exclusion. Three independent replicates of the experiment were performed. 2.2.2. Cell viability Cell viability within the microcarrier culture was assessed every 2–3 days using the fluorescin diacetate (FDA, Sigma) and propidium iodide (PI, Sigma) paravital staining method as previously described [24]. In short, 0.5 ml samples were taken from the microcarrier spinner cultures. After rinsing with PBS, 1 ml of dye solution (final concentration FDA: 0.67 mg/ml, PI: 5 mg/ml) was added. Samples were placed in the dark for 5 min and subsequently rinsed with PBS - 6 times to eliminate background fluorescence. Samples were transferred to slides and visualized immediately using a
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microscope (E600, Nikon) with a double filter block (dichroic mirror 505 and 590 nm). Additionally, 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) staining was used to monitor cell viability at days 3 and 7. Samples (0.5 ml) from spinner microcarrier cultures were rinsed with PBS and 200 ml of 1 mg/ml MTT (Sigma-Aldrich) dissolved in DMEM without phenol red (Gibco-BRL) was added. After 15 min incubation at 37 C samples were screened using a light microscope (E600, Nikon). 2.2.3. T-flask expansion Isolated chondrocytes were plated either in 25 or 300 cm2 T-flasks at 1000 cells/cm2 in medium 1 and maintained at 37 C in a humidified 5% CO2 incubator. In order to monitor cell proliferation, chondrocytes were detached from two flasks daily using 0.25% Trypsin/1.0 mm EDTA and counted using a particle count and size analyser (Coulter Corporation). 2.3. In-vitro redifferentiation 2.3.1. Pellet preparation and culture Suspensions of chondrocytes were transferred into a 12-ml polypropylene Falcon centrifuge tube (500,000 cells/tube) and centrifuged for 4 min at 300g. The resulting pellets were statically cultured for 7 days at 37 C in a humidified 5% CO2 incubator in either medium 1, medium 2 [2](DMEM-Glutamax-1 supplemented with 1% non-essential amino acids, 100 units/ml penicillin/100 mg/ml streptomycin, 10% FBS, 10 mg/ml insulin (Sigma-Aldrich), 50 mg/ml ascorbic acid), or medium 3 [25](HEPES-buffered DMEM-Glutamax-1 (Gibco-BRL), supplemented with 0.2 mm ascorbic acid 2-phosphate, 1 ITS+1 (Sigma-Aldrich), 10 ng/ml TGF-b2 (R&D Systems), 10 ng/ml IGF-1 (R&D Systems), 100 units/ml penicillin, and 100 mg/ml streptomycin). Medium was refreshed every 2–3 days for the course of the culture period. 2.3.2. Cell–polymer construct preparation Porous blocks were produced with a hot press and salt leaching method, as previously described [26]. In brief, 300PEGT55PBT45 (PolyActivet, IsoTis N.V.) powders (PEGT/PBT weight ratio of 55/45 and a PEGT molecular weight of 300 g/mol) with particle size of less than 600 mm were homogeneously mixed with sodium chloride grains. The grain size was 500–600 mm and the amount of the salt was adjusted to a final volume percentage of 80%. The mixture was compression moulded into a block, and the block was subsequently immersed in demineralized water to remove the sodium chloride. Cylindrical scaffolds (+ 4 mm) were cored out of the 4 mm thick porous blocks.
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2.3.3. Cell–polymer construct culture Scaffolds were steam-sterilized (15 min, 121 C) and incubated at least 1 h in medium 2 prior cell seeding. Chondrocytes from either T-flasks or microcarrier culture were dynamically seeded onto cylindrical scaffolds (3.0 106 cells per scaffold) fixed to needles in spinner flasks [27] at 60 rpm at 37 C in a humidified 5% CO2 incubator. Constructs were cultured up to 14 days in medium 2. Culture medium was replaced every 2–3 days during culture.
Aldrich) [28]. Intensity of color change was quantified immediately in a microplate reader (EL 312e Bio-TEK Instruments) by measuring absorbance at 520 nm. Values were compared to those from a standard of cystein–HCl. Hydroxyproline content: Hydroxyproline was determined as a measure of total collagen present in each construct. Aliquots of proteinase K digests were hydrolyzed in 6 n HCl at 110 C for 16 h. The resulting solution was assayed for hydroxyproline using methods that have been described in detail elsewhere [29].
2.4. In vivo redifferentiation 2.6. Statistics After 14 days of culture as described above, the resulting chondrocyte-polymer constructs were implanted in subcutaneous pockets of 6 week old nude mice (4 scaffolds/animal; HdCpb:NMRI-nu, Harlan, The Netherlands). The mice were killed two weeks after implantation and the constructs were analyzed histologically and biochemically (see following).
Statistical significance was assessed by analysis of variance (ANOVA) followed by Tukey’s posthoc test with Sigma Stat (SPSS Inc.) using po0:05 as the criteria for statistical significance.
3. Results 2.5. Analysis 3.1. Expansion 2.5.1. Histological analysis Samples of pellet cultures were taken after 7 days of culture. Samples of scaffold cultures were removed after 3 days (3d), and 14 days (14d) of in vitro culture and after 14 days in vitro and an additional 14 days in vivo (14d+14d). Samples were fixed using 0.14 m cacodylate buffer (pH=7.2–7.4)/1.5% glutaraldehyde (Merck), dehydrated, embedded in glycol methacrylate (Merck) and cut to yield 5 mm thick sections. Sections were stained with haematoxylin (Sigma-Aldrich) and fast green (Merck) for cells and with safranin-O (SigmaAldrich) for glycosaminoglycans (GAG). Samples were then visualized using a light microscope (E600, Nikon). 2.5.2. Biochemical analysis Construct samples (n ¼ 3 per data point) for biochemical quantification of DNA, GAG and hydroxyproline content were removed after 3 days (3d), and 14 days (14d) of in vitro culture and after 14 days in vitro and an additional 14 days in vivo (14d+14d). Constructs were placed in a solution containing proteinase K (1 mg/ml), pepstatin A (10 mg/ml) and iodoacetamide (185 mg/ml) (Sigma-Aldrich) overnight at 56 C to digest the cells and extra-cellular matrix (ECM) formed during culture. These samples were then evaluated for cell number, GAG and total collagen as follows: DNA content: Quantification of total DNA was performed with a Cyquant dye kit (Molecular Probes) as per manufacturer’s instructions using a fluorescent plate reader (Perkin Elmer). GAG content: GAG was quantitatively determined by reaction with dimethylmethylene blue dye (Sigma-
The intermittent stirring regime yielded a relatively homogeneous cell distribution between carriers within the culture as assessed after 3 days by MTT staining (Fig. 2A). During culture, cells remained viable, as demonstrated by MTT and FDA/PI staining (Figs. 2B and C). Furthermore, cells harvested after 12 days of expansion were viable as indicated by exclusion of trypan blue dye. In addition, cell number increased over time, as demonstrated qualitatively by MTT staining after 3 and 7 days of culture (Figs. 2A and B). For human nasal chondrocytes cultured on macroporous microcarriers, a doubling time of 1.0770.14 days was observed. This doubling time was within the same range as doubling times observed in parallel T-flask cultures (1.2070.36 days). The carrier cultures, initially seeded with approximately 10 cells per carrier (1 106 cells/100.000 carriers), yielded 400–500 cells per carrier after the 12day expansion phase. 3.2. Pellet redifferentiation in vitro Regardless of expansion method, cell pellets showed no staining for GAG after 7 days in medium 1 (Figs. 3A and D). In insulin-containing medium (2), only carrierexpanded cells demonstrated chondrocyte-like morphology and staining for GAG (Figs. 3B and E). With serum free medium (3) supplemented with growth factors, GAG production in pellets was evident irrespective of expansion method (Figs. 3C and F). However, based on cell shape and GAG staining, pellets of microcarrier-expanded cells demonstrated a higher degree of
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A (A)
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Fig. 3. Proteoglycan staining (safranin-O) of cell pellets from T-flask(A,B,C) and microcarrier- (D,E,F) expanded nasal chondrocytes, cultured for 7 days in medium 1 (A,D), 2 (B,E), and 3 (C,F). Original magnification 40 .
(C) Fig. 2. MTT (A,B) and FDA/PI (C) assay of nasal chondrocytes cultured on CultiSpher G carriers for 3 days (A) and 7 days (B,C). Bar=400 mm.
redifferentiation, whilst pellets of T-flask expanded cells had a more fibrous appearance and stained less intense for GAG.
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3.3. Scaffold redifferentiation in vitro and in vivo After 3 days of culture cells had spread throughout the entire scaffold. Nevertheless, more cells were found within the periphery of the constructs (results not shown). After 14 days of culture localized areas with intense staining for GAG were observed within both constructs (Fig. 4). In addition, a layer of elongated cells was present within the highly cellular outside edge. The DNA assay demonstrated that DNA content gradually increased with time, and no significant
(B) Fig. 4. Proteoglycan staining (safranin-O) of constructs, from either microcarrier- (A) or T-flask- (B) expanded chondrocytes, cultured for 14 days. Original magnification 100 , arrow indicates the polymer scaffold.
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Fig. 6. Proteoglycan staining (safranin-O) of constructs, from either microcarrier- (A) or T-flask- (B) expanded chondrocytes, cultured for 14 days and, subsequently, subcutaneously implanted in nude mice for 14 days. Original magnification 40 , arrow indicates the polymer scaffold.
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Fig. 5. Quantification of DNA (A), glycosaminoglycans (B) and hydroxyproline (C) per tissue engineered construct cultured for either 3 days (3d) or 14 days (14d) in medium 2 or for 14 days in medium 2 and, subsequently, subcutaneously implanted for 14 days in nude mice (14d+14d). (DNA, glycosaminoglycan and hydroxyproline content are expressed as mean7SD and po0:05 was considered significant. =significantly different.)
differences were found between either of the two expansion methods (Fig. 5A). GAG content per scaffold increased from approximately 30 mg at day 3–130 mg at day 14 in vitro. At the in vitro time points, pellets from microcarrier or twodimensionally expanded cells showed no significant difference in total GAG content. After in vivo implantation, GAG content was found significantly higher in ‘14d+14d’ constructs from microcarrier expanded cells (288.5729.2 mg) than in constructs from twodimensionally expanded cells (164.0728.7 mg) (Fig. 5B). These observations correlate with the safranin-O staining at these time points (Fig. 4). At the 14d+14d time point, intense staining for GAG was localized in both constructs (Fig. 6). Nevertheless, more regions demonstrating round cells in lacunae, an indicator of a cartilage-like morphology, were observed within the constructs from microcarrier-expanded cells. Further-
more, more staining for GAG was localized within the outer regions of these constructs (Fig. 6B). Although, total collagen, as measured by hydroxyproline content, did not increase dramatically during 14 days of in vitro culture (Fig. 5C), constructs from Tflask-expanded cells produced slightly more collagen at this time point (not significant). After subcutaneous implantation of the constructs, an increase in collagen content was observed for both conditions. The amount of collagen produced by chondrocytes expanded on microcarriers after 14 days of implantation was slightly higher; however, the difference was not significant.
4. Discussion These results demonstrate that isolated human nasal chondrocytes can be propagated on CultiSpher microcarriers to yield viable cells and comparable expansion rates to T-flask culture. Previous investigations have emphasized that the initial phase of a microcarrier culture is usually the most critical stage, as non-attached or inhomogenously distributed cells have considerable effects on the yield and growth rate obtained [30]. Additionally, it has been reported that mammalian cells, in general, need more time to attach to CultiSpher G microcarriers in comparison to other commonly used microcarriers, such as Cytodex [11]. Therefore, a 24-h
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period of intermittent stirring was applied to assure homogeneous distribution of attached cells throughout the microcarrier culture. Doubling times, obtained in this system (1.0770.14 days) were lower than reported previously for human nasal chondrocytes on microcarriers (74 days) [7]. In vitro redifferentiation of human nasal septum chondrocytes in pellets was performed using three culture media. Medium 1, which has been used by several authors for 3-dimensional bovine chondrocyte culture [31,32], did not yield positive staining for proteoglycans at the time point evaluated (7 days), regardless of the expansion method (Fig. 2). However, pellet cultures in media 2 and 3 demonstrated that redifferentiation (GAG production) was enhanced by microcarrier expansion. These media contain supplements previously shown to stimulate chondrogenesis [2,25]. From these results, an evident question is how culture on microcarriers stimulates the subsequent redifferentiation. The stimulatory effects observed could be related to several specific aspects of the microcarrier culture, including (I) fluid-induced shear, (II) better reproduction of their natural three-dimensional environment and (III) the presence of nutrient gradients, as explained below in greater detail. It has been suggested that the macroporous structure of CultiSpher microcarriers creates a protected environment for the chondrocytes, as the cells within the interior will experience lower shear forces than those encountered with non-porous carriers [11,33]. This protected environment could explain why considerable growth rates were observed. On the other hand, cells proliferating on the outside of the carrier experience shear forces, as do chondrocytes in natural cartilage [34]. Smith and colleagues [34,35] reported stimulatory effects of shear stress applied in vitro on chondrogenesis. More specifically, Frondoza et al. [6] and Bouchet et al. [7] observed enhanced expression of chondrocytespecific proteins during spinner-flask culture with nonporous microcarriers and suggested that articular chondrocytes benefit from a mechanically active tissue culture environment. An alternative explanation for the increase in chondrogenic potential with microcarrier-expanded cells is related to the microenvironment experienced by the cells during the expansion phase. Chondrocytes in vivo are surrounded by their ECM, composed of mainly collagen type II and GAG. Culturing isolated chondrocytes in a three-dimensional gelatin-based carrier more closely mimics the in vivo situation. The porous structure allows attachment in three dimensions and, in addition, the gelatin has, like cartilage, hydrogel-like properties [36,37]. Furthermore, the denatured collagen structure could still contain sequences that can bind to the cell via integrin or other receptors [38]. This could
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contribute to the enhancement of the chondrocytic phenotype during expansion. As a result, increased GAG production, a marker of differentiated chondrocytes, can be observed when cells are, subsequently, seeded in pellets or polymer scaffolds. The enhanced redifferentiation potential of microcarrier-expanded chondrocytes could also be related to nutrient gradients. As a result of multiple cell layers within the macroporous CultiSpher microcarriers, gradients of nutrients, such as oxygen, are likely to develop [39]. In particular, oxygen has been shown to be a controlling agent of developmental processes, such as chondrogenesis [40]. Additionally, several studies have reported that low oxygen tensions stimulate the production of hyaline cartilage specific collagen type II and proteoglycans in vitro [41–44]. Because the distinct morphology and metabolism of chondrocytes depends upon their position in the cartilage tissue, Grimshaw and Mason [45] hypothesized that this may be, at least in part, due to the position of the chondrocyte within the oxygen gradient. Results from pellet cultures after T-flask and microcarrier expansion, support the concept that supplements such as TGF-b2 improve neo-cartilage formation from cells in vitro, as assessed by safranin-O staining. However, such growth factors can be costly and, in some cases, unnecessary. Therefore, in subsequent experiments in which chondrocytes were cultured on PEGT/PBT scaffolds, only medium 2 was used, as it contained a minimum amount of supplements (insulin only), while still showing evidence of cartilage matrix production, as was assessed by GAG formation, after 7 days (Fig. 3E). In the in vitro and following in vivo study, scaffold cultures demonstrated that expanded chondrocytes maintained the ability to differentiate (Figs. 4 and 6). It was also found that microcarrierexpanded chondrocytes produce significantly more GAG after 14 days in vitro followed by 14 days in vivo than two-dimensionally expanded cells. Staining for GAG was not homogeneously distributed, which is likely due to the presence of the non-degraded scaffold. The enhanced production of GAG, a cartilage marker, suggests that microcarrier culture aids expression of the chondrocytic phenotype. Surprisingly, total collagen production after 14 days in vitro culture was higher, although not significant, in constructs from T-flaskexpanded cells. After in vivo culture collagen content increased for both samples, but no significant differences between expansion methods were found. However, the assay used quantifies total collagen produced and cannot distinguish between collagen types. Therefore, quantification of hyaline-specific collagen type II within the constructs was not possible. Previous investigations have shown the potential of macroporous carriers as a delivery system for human adult mesenchymal stem cells for tissue engineering
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applications, such as bone repair [46]. Furthermore, nasal chondrocytes have been shown to be a promising cell source for autologous tissue engineering approaches for the repair of cartilage defects. Increased hyaline cartilage-like tissue formation from nasal chondrocytes over articular chondrocytes, has been reported [2,3]. Our experiments have demonstrated that such carriers are also potent matrices for nasal septum chondrocyte expansion, while simultaneously promoting post-expansion chondrogenesis. Nasal cartilage is subjected to tensile stresses due to the interaction of the collagen network and the swelling pressure generated by proteoglycan–ion interactions [47]. However, unlike articular cartilage, it is non-loadbearing, which may influence how nasal septum chondrocytes behave in a loaded site. Therefore, further research on the behavior of nasal chondrocytes and their extra-cellular matrix in a mechanically loaded environment is essential before they can be used to treat articular defects. With human nasal chondrocytes proliferated on macroporous gelatin-based microcarriers, reparative cells, capable of synthesizing a tissue with a cartilagelike appearance, can be obtained. Although the exact mechanism by which culture on microcarriers enhances the chondrocytic phenotype remains uncertain, combining these cells with a scaffold made from a biocompatible and biodegradable PEGT/PBT material, holds promise for future clinical use in repair of articular cartilage defects.
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[9] Baker TL, Goodwin TJ. Three-dimensional culture of bovine chondrocytes in rotating-wall vessels. In Vitro Cell Dev Biol Anim 1997;33:358–65. [10] Nilsson K. Microcarrier cell culture. Biotechnol Genet Eng Rev 1988;6:403–39. [11] Ng YC, Berry JM, Butler M. Optimization of physical parameters for cell attachment on macroporous microcarriers. Biotech Bioeng 1996;50:627–35. [12] Temenoff JS, Mikos AG. Injectable biodegradable materials for orthopedic tissue engineering. Biomaterials 2000;21:2405–12. [13] Beumer GJ, van Blitterswijk CA, Ponec M. Biocompatibility of a biodegradable matrix used as a skin substitute: an in vivo evaluation. J Biomed Mater Res 1994;28:545–52. [14] Beumer GJ, van Blitterswijk CA, Bakker D, Ponec M. Cellseeding and in vitro biocompatibility evaluation of polymeric matrices of PEO/PBT copolymers and PLLA. Biomaterials 1993;14:598–604. [15] Radder AM, Davies JE, Leenders H, van Blitterswijk CA. Interfacial behavior of PEO/PBT copolymers (Polyactive) in a calvarial system: an in vitro study. J Biomed Mater Res 1994;28:269–77. [16] Radder AM, Leenders H, van Blitterswijk CA. Interface reactions to PEO/PBT copolymers (Polyactive) after implantation in cortical bone. J Biomed Mater Res 1994;28:141–51. [17] Meijer GJ, van Dooren A, Gaillard ML, Dalmeijer R, de Putter C, Koole R, van Blitterswijk CA. Polyactive as a bone-filler in a beagle dog model. Int J Oral Maxillofac Surg 1996;25: 210–6. [18] Grote JJ, Bakker D, Hesseling SC, van Blitterswijk CA. New alloplastic tympanic membrane material. Am J Otol 1991;12: 329–35. [19] Bakker D, van Blitterswijk CA, Hesseling SC, Koerten HK, Kuijpers W. Grote JJ Biocompatibility of a polyether urethane, polypropylene oxide, and a polyether polyester copolymer. A qualitative and quantitative study of three alloplastic tympanic membrane materials in the rat middle ear. J Biomed Mater Res 1990;24:489–515. [20] van Blitterswijk CA, van den Brink J, Leenders H, Bakker D. The effect of PEO ratio on degradation, calcification and bonebonding of PEO/PBT copolymer (Polyactive). Cells Mater 1993;3:23–36. [21] Sakkers RJ, de Wijn JR, Dalmeijer R, Brand R, van Blitterswijk CA. Evaluation of copolymers of polyethylene oxide and polybuthylene terephtalate (Polyactive): mechanical behaviour. Mater Med 1998;9:375–9. [22] Woodfield TBF, Bezemer JM, Pieper JS, van Blitterswijk CA, Riesle J. Scaffolds for tissue engineering of cartilage. Crit Rev Euk Gene Exp 2002;12:207–35. [23] Kato Y, Iwamoto M, Koike T, Suzuki F, Takano Y. Terminal differentiation and calcification in rabbit chondrocyte cultures grown in centrifuge tubes: regulation by transforming growth factor beta and serum factors. Proc Natl Acad Sci USA 1988;85:9552–6. [24] Clements KM, Bee ZC, Crossingham GV, Adams MA, Sharif M. How severe must repetitive loading be to kill chondrocytes in articular cartilage? Osteoarthr Cartilage 2001;9:499–507. [25] Yaeger PC, Masi TL, de Ortiz JL, Binette F, Tubo R, McPherson JM. Synergistic action of transforming growth factor-beta and insulin-like growth factor-I induces expression of type II collagen and aggrecan genes in adult human articular chondrocytes. Exp Cell Res 1997;237:318–25. [26] Du C, Klasens P, Haan RE, Bezemer J, Cui FZ, de Groot K, Layrolle P. Biomimetic calcium phosphate coatings on PolyActive 1000/70/30. J Biomed Mater Res 2002;59:535–46. [27] Freed LE, Vunjak-Novakovic G. Microgravity tissue engineering. In Vitro Cell Dev Biol Anim 1997;33:381–5.
ARTICLE IN PRESS J. Malda et al. / Biomaterials 24 (2003) 5153–5161 [28] Farndale RW, Buttle DJ, Barrett AJ. Improved quantitation and discrimination of sulphated glycosaminoglycans by use of dimethylmethylene blue. Biochim Biophys Acta 1986;883:173–7. [29] Morales TI, Woessner JF, Howell DS, Marsh JM, Le Maire WJ. A microassay for the direct demonstration of collagenolytic activity in Graafian follicles of the rat. Biochim Biophys Acta 1978;524:428–34. [30] Gebb C, Lundgren B, Clark J, Lindskog U. Harvesting and subculturing cells growing on denatured-collagen coated microcarriers. Dev Biol Stand 1984;55:57–62. [31] Freed LE, Hollander AP, Martin I, Barry JR, Langer R, VunjakNovakovic G. Chondrogenesis in a cell–polymer–bioreactor system. Exp Cell Res 1998;240:58–65. [32] Martin I, Obradovic B, Freed LE, Vunjak-Novakovic G. Method for quantitative analysis of glycosaminoglycan distribution in cultured natural and engineered cartilage. Ann Biomed Eng 1999;27:656–62. [33] Moran E. A microcarrier-based cell culture process for the production of a bovine respiratory syncytial virus vaccine. Cytotechnology 1999;29:135–48. [34] Smith RL, Donlon BS, Gupta MK, Mohtai M, Das P, Carter DR, Cook J, Gibbons G, Hutchinson N, Schurman DJ. Effects of fluid-induced shear on articular chondrocyte morphology and metabolism in vitro. J Orthop Res 1995;13:824–31. [35] Smith RL, Trindade MC, Ikenoue T, Mohtai M, Das P, Carter DR, Goodman SB, Schurman DJ. Effects of shear stress on articular chondrocyte metabolism. Biorheology 2000;37:95–107. [36] Kang HW, Tabata Y, Ikada Y. Fabrication of porous gelatin scaffolds for tissue engineering. Biomaterials 1999;20:1339–44. [37] Tabata Y, Ikada Y. Vascularization effect of basic fibroblast growth factor released from gelatin hydrogels with different biodegradabilities. Biomaterials 1999;20:2169–75.
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Expansion of human nasal chondrocytes on macroporous microcarriers enhances redifferentiation J. Maldaa,b,*, E. Kreijvelda, J.S. Temenoffc, C.A. van Blitterswijka,b, J. Rieslea b
a IsoTis N.V., P.O. Box 98, 3720 AB Bilthoven, The Netherlands Institute for BioMedical Technology (BMTI), University of Twente, P.O. Box 217, 7500 AE Enschede, The Netherlands c Department of Bioengineering, Rice University, Houston, TX 77251-1892, USA
Received 9 December 2002; accepted 23 May 2003
Abstract Articular cartilage has a limited capacity for self-repair. To overcome this problem, it is expected that functional cartilage replacements can be created from expanded chondrocytes seeded in biodegradable scaffolds. Expansion of chondrocytes in twodimensional culture systems often results in dedifferentiation. This investigation focuses on the post-expansion phenotype of human nasal chondrocytes expanded on macroporous gelatin CultiSpher G microcarriers. Redifferentiation was evaluated in vitro via pellet cultures in three different culture media. Furthermore, the chondrogenic potential of expanded cells seeded in polyethylene glycol terephthalate/ polybuthylene terephthalate (PEGT/PBT) scaffolds, cultured for 14 days in vitro, and subsequently implanted subcutaneously in nude mice, was assessed. Chondrocytes remained viable during microcarrier culture and yielded doubling times (1.0770.14 days) comparable to T-flask expansion (1.2070.36 days). Safranin-O staining from pellet culture in different media demonstrated that production of GAG per cell was enhanced by microcarrier expansion. Chondrocyte–polymer constructs with cells expanded on microcarriers contained significantly more proteoglycans after subcutaneous implantation (288.5729.2 mg) than those with T-flask-expanded cells (164.0728.7 mg). Total collagen content was similar between the two groups. This study suggests that macroporous gelatin microcarriers are effective matrices for nasal chondrocyte expansion, while maintaining the ability of chondrocyte differentiation. Although the exact mechanism by which chondrocyte redifferentiation is induced through microcarrier expansion has not yet been elucidated, this technique shows promise for cartilage tissue engineering approaches. r 2003 Elsevier Ltd. All rights reserved. Keywords: Nasal chondrocytes; Redifferentiation; Polyethylene glycol terephthalate/polybuthylene terephthalate (PEGT/PBT); Macroporous microcarriers; Insulin; Tissue engineering
1. Introduction Because of the limited self-renewal capability of articular cartilage, an estimated one million people per year require treatment of cartilage defects [1]. Once damaged, these defects do not heal spontaneously. Therefore, tissue engineering approaches offer new possibilities for functional and structural restoration of the damaged or lost tissue. In the development of a tissue engineered implant for the treatment of articular cartilage defects several requirements should be considered. In order to functionally and mechanically restore the defect, the implant *Corresponding author. 0142-9612/$ - see front matter r 2003 Elsevier Ltd. All rights reserved. doi:10.1016/S0142-9612(03)00428-9
should include reparative cells capable of synthesising hyaline cartilage-specific extra-cellular matrix (ECM), supported by a biodegradable scaffold that matches the mechanical properties of the surrounding tissue. While a variety of cell sources may be used in the construct, an autologous patient biopsy can be preferable as this decreases the risk of immunogenic response and disease transfer. Articular cartilage from little loadbearing regions could be used, but, due to donor site morbidity, the amount of tissue is limited. Therefore, the use of alternative autologous cell sources, such as human septum cartilage, has been evaluated [2,3]. Harvest of cartilage from the nasal septum requires less invasive surgery then harvest from articular sources and the resulting morbidity is minimal [2]. Furthermore,
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Fig. 1. Experimental design. Isolated human nasal chondrocytes were expanded for 12 days in T-flasks or on CultiSpher G microcarriers. To evaluate the post-expansion chondrocytic phenotype, the resulting cells were seeded either in pellets or on PEGT/PBT scaffolds and cultured for 7 and 14 days, respectively. Furthermore, PEGT/PBT-cartilage constructs were implanted for an additional 14 days subcutaneously (s.c.) in nude mice.
several investigators have reported increased cartilage formation by nasal septum chondrocytes in comparison to articular chondrocytes and indicated the potential of nasal cartilage as a cell source for cartilage tissue engineering strategies [2,3]. To reduce the size of the required biopsy, prior to seeding on scaffolds, cells must be expanded to obtain a sufficient cell number. During this process, the chondrocytes loose their spherical shape and acquire a fibroblast-like appearance [4,5]. Accordingly, expression of hyaline cartilage markers, aggrecan and collagen type II, decreases, while expression of non-hyaline cartilage specific collagen type I increases [5]. In an effort to overcome this problem, the culture of chondrocytes on microcarriers, has been investigated as a means to maintain a chondrocytic phenotype, resulting in specific expression of collagen type II, aggrecan and beta-1 integrin [6–9]. Additionally, we recently demonstrated that expansion of immature bovine chondrocytes on non-porous Cytodex 1 microcarriers increased neo-cartilage formation in subsequent pellet cultures, as compared to pellets of chondrocytes expanded in T-flasks (Malda et al., in press). Macroporous microcarriers, including gelatin-based carriers, offer the advantage that the surface area per bead can be further increased, thus allowing the attachment of more cells per carrier [10,11]. The use of gelatin-based macroporous microcarriers is also advantageous in that, since the beads are susceptible to proteolytic enzymes, such as trypsin, harvest of expanded cells is greatly simplified. Therefore, in this study, human septal chondrocytes were cultured on marcoporous CultiSpher G gelatin microspheres prior to phenotypic analysis. In addition to reparative cells, the scaffold plays a pivotal role in cartilage tissue engineering. It serves as a template to guide the growth and organization of the
developing tissue and gives stability to the neo-tissue [12]. Our method adopts a porous biodegradable segmented-block-copolymer scaffold. The biocompatibility of this material, which is composed of polyethylene glycol terephthalate (PEGT) and polybutylene terephthalate (PBT) segments, has been demonstrated in several studies [13–20]. By varying the ratio of the alternating soft (PEGT) and hard (PBT) segments the mechanical properties of this material can be tailored [21] to match the mechanical properties of cartilage [22]. In the present studies, the post-expansion phenotype of human nasal chondrocytes expanded on macroporous gelatin CultiSpher G microcarriers was investigated (Fig. 1). Neo-cartilage formation was evaluated in vitro via pellet culture [23] in three different media. Furthermore, the potential of expanded cells was assessed after seeding in PEGT/PBT copolymer scaffold culture for 14 days in vitro, and subsequent subcutaneous implantation in nude mice. Proteoglycan production in the cellpolymer constructs was evaluated histologically via safranin-O staining at various time points, while proteoglycan and total collagen production were quantified concurrently using biochemical assays.
2. Materials and methods 2.1. Cell isolation Nasal cartilage was obtained from patients (n ¼ 9; age range: 18–51 years, average: 36 years) undergoing nasal septum reconstruction. Cartilage was dissected in approximately 1-mm3 cubes. Chondrocytes were isolated by overnight digestion with 0.15% type II collagenase (Worthington Biochemical) at 37 C. After three washes in phosphate-buffered saline (PBS)
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(Gibco-BRL), cells were suspended in medium 1 (HEPES-buffered DMEM (Gibco-BRL) supplemented with 10% FCS (Sigma-Aldrich), 0.2 mm ascorbic acid 2phosphate (Gibco-BRL), 0.1 mm non-essential amino acids (Sigma-Aldrich), 0.4 mm proline (Sigma-Aldrich), 100 units/ml penicillin (Gibco-BRL), and 100 mg/ml streptomycin (Gibco-BRL) and either seeded in T-flasks or microcarrier culture. 2.2. Cell expansion 2.2.1. Microcarrier seeding and expansion Macroporous gelatin-based CultiSpher G microcarrier beads (Percell Biolytica), + 130–180 mm, were prepared according to the manufacturer’s instructions. In short, microcarriers were hydrated prior to use in PBS (Gibco-BRL) at 37 C and steam-sterilized (15 min, 121 C) in Sigmacote (Sigma-Aldrich) treated bottles. Microcarriers were then rinsed twice with PBS and once with 37 C medium 1 and transferred to a Sigmacote treated 250 ml spinner flask (working volume 100 ml, end concentration 1 mg CultiSpher G/ml). Microcarriers were cell-seeded (1.0 106 cells/flask) in 50 ml of medium 1 using an intermittent stirring regime (30 min at 0 rpm, 30 s at 20 rpm) for 24 h (n=2). After seeding, 50 ml of medium 1 was added and the stirring speed was increased to 50 rpm. All cultures were maintained at 37 C in a humidified 5% CO2 incubator. For daily cell counts, 0.5 ml samples were rinsed with PBS and 150 ml 0.25% Trypsin/1.0 mm EDTA (Gibco-BRL) was added to dissolve the microcarriers. After 15 min of incubation at 37 C, cells were counted using a particle count and size analyser (Coulter Corporation). For cell harvesting, microcarriers were allowed to settle for 10 min. The supernatant was removed and the culture was rinsed twice with PBS. Subsequently, 15 ml 0.25% Trypsin/1.0 mm EDTA was added to dissolve the microcarriers. After 15 min incubation at 37 C, 15 ml of culture medium was added and cells were counted using a particle count and size analyser (Coulter Corporation). Cell viability was measured using trypan blue (SigmaAldrich) exclusion. Three independent replicates of the experiment were performed. 2.2.2. Cell viability Cell viability within the microcarrier culture was assessed every 2–3 days using the fluorescin diacetate (FDA, Sigma) and propidium iodide (PI, Sigma) paravital staining method as previously described [24]. In short, 0.5 ml samples were taken from the microcarrier spinner cultures. After rinsing with PBS, 1 ml of dye solution (final concentration FDA: 0.67 mg/ml, PI: 5 mg/ml) was added. Samples were placed in the dark for 5 min and subsequently rinsed with PBS - 6 times to eliminate background fluorescence. Samples were transferred to slides and visualized immediately using a
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microscope (E600, Nikon) with a double filter block (dichroic mirror 505 and 590 nm). Additionally, 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) staining was used to monitor cell viability at days 3 and 7. Samples (0.5 ml) from spinner microcarrier cultures were rinsed with PBS and 200 ml of 1 mg/ml MTT (Sigma-Aldrich) dissolved in DMEM without phenol red (Gibco-BRL) was added. After 15 min incubation at 37 C samples were screened using a light microscope (E600, Nikon). 2.2.3. T-flask expansion Isolated chondrocytes were plated either in 25 or 300 cm2 T-flasks at 1000 cells/cm2 in medium 1 and maintained at 37 C in a humidified 5% CO2 incubator. In order to monitor cell proliferation, chondrocytes were detached from two flasks daily using 0.25% Trypsin/1.0 mm EDTA and counted using a particle count and size analyser (Coulter Corporation). 2.3. In-vitro redifferentiation 2.3.1. Pellet preparation and culture Suspensions of chondrocytes were transferred into a 12-ml polypropylene Falcon centrifuge tube (500,000 cells/tube) and centrifuged for 4 min at 300g. The resulting pellets were statically cultured for 7 days at 37 C in a humidified 5% CO2 incubator in either medium 1, medium 2 [2](DMEM-Glutamax-1 supplemented with 1% non-essential amino acids, 100 units/ml penicillin/100 mg/ml streptomycin, 10% FBS, 10 mg/ml insulin (Sigma-Aldrich), 50 mg/ml ascorbic acid), or medium 3 [25](HEPES-buffered DMEM-Glutamax-1 (Gibco-BRL), supplemented with 0.2 mm ascorbic acid 2-phosphate, 1 ITS+1 (Sigma-Aldrich), 10 ng/ml TGF-b2 (R&D Systems), 10 ng/ml IGF-1 (R&D Systems), 100 units/ml penicillin, and 100 mg/ml streptomycin). Medium was refreshed every 2–3 days for the course of the culture period. 2.3.2. Cell–polymer construct preparation Porous blocks were produced with a hot press and salt leaching method, as previously described [26]. In brief, 300PEGT55PBT45 (PolyActivet, IsoTis N.V.) powders (PEGT/PBT weight ratio of 55/45 and a PEGT molecular weight of 300 g/mol) with particle size of less than 600 mm were homogeneously mixed with sodium chloride grains. The grain size was 500–600 mm and the amount of the salt was adjusted to a final volume percentage of 80%. The mixture was compression moulded into a block, and the block was subsequently immersed in demineralized water to remove the sodium chloride. Cylindrical scaffolds (+ 4 mm) were cored out of the 4 mm thick porous blocks.
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2.3.3. Cell–polymer construct culture Scaffolds were steam-sterilized (15 min, 121 C) and incubated at least 1 h in medium 2 prior cell seeding. Chondrocytes from either T-flasks or microcarrier culture were dynamically seeded onto cylindrical scaffolds (3.0 106 cells per scaffold) fixed to needles in spinner flasks [27] at 60 rpm at 37 C in a humidified 5% CO2 incubator. Constructs were cultured up to 14 days in medium 2. Culture medium was replaced every 2–3 days during culture.
Aldrich) [28]. Intensity of color change was quantified immediately in a microplate reader (EL 312e Bio-TEK Instruments) by measuring absorbance at 520 nm. Values were compared to those from a standard of cystein–HCl. Hydroxyproline content: Hydroxyproline was determined as a measure of total collagen present in each construct. Aliquots of proteinase K digests were hydrolyzed in 6 n HCl at 110 C for 16 h. The resulting solution was assayed for hydroxyproline using methods that have been described in detail elsewhere [29].
2.4. In vivo redifferentiation 2.6. Statistics After 14 days of culture as described above, the resulting chondrocyte-polymer constructs were implanted in subcutaneous pockets of 6 week old nude mice (4 scaffolds/animal; HdCpb:NMRI-nu, Harlan, The Netherlands). The mice were killed two weeks after implantation and the constructs were analyzed histologically and biochemically (see following).
Statistical significance was assessed by analysis of variance (ANOVA) followed by Tukey’s posthoc test with Sigma Stat (SPSS Inc.) using po0:05 as the criteria for statistical significance.
3. Results 2.5. Analysis 3.1. Expansion 2.5.1. Histological analysis Samples of pellet cultures were taken after 7 days of culture. Samples of scaffold cultures were removed after 3 days (3d), and 14 days (14d) of in vitro culture and after 14 days in vitro and an additional 14 days in vivo (14d+14d). Samples were fixed using 0.14 m cacodylate buffer (pH=7.2–7.4)/1.5% glutaraldehyde (Merck), dehydrated, embedded in glycol methacrylate (Merck) and cut to yield 5 mm thick sections. Sections were stained with haematoxylin (Sigma-Aldrich) and fast green (Merck) for cells and with safranin-O (SigmaAldrich) for glycosaminoglycans (GAG). Samples were then visualized using a light microscope (E600, Nikon). 2.5.2. Biochemical analysis Construct samples (n ¼ 3 per data point) for biochemical quantification of DNA, GAG and hydroxyproline content were removed after 3 days (3d), and 14 days (14d) of in vitro culture and after 14 days in vitro and an additional 14 days in vivo (14d+14d). Constructs were placed in a solution containing proteinase K (1 mg/ml), pepstatin A (10 mg/ml) and iodoacetamide (185 mg/ml) (Sigma-Aldrich) overnight at 56 C to digest the cells and extra-cellular matrix (ECM) formed during culture. These samples were then evaluated for cell number, GAG and total collagen as follows: DNA content: Quantification of total DNA was performed with a Cyquant dye kit (Molecular Probes) as per manufacturer’s instructions using a fluorescent plate reader (Perkin Elmer). GAG content: GAG was quantitatively determined by reaction with dimethylmethylene blue dye (Sigma-
The intermittent stirring regime yielded a relatively homogeneous cell distribution between carriers within the culture as assessed after 3 days by MTT staining (Fig. 2A). During culture, cells remained viable, as demonstrated by MTT and FDA/PI staining (Figs. 2B and C). Furthermore, cells harvested after 12 days of expansion were viable as indicated by exclusion of trypan blue dye. In addition, cell number increased over time, as demonstrated qualitatively by MTT staining after 3 and 7 days of culture (Figs. 2A and B). For human nasal chondrocytes cultured on macroporous microcarriers, a doubling time of 1.0770.14 days was observed. This doubling time was within the same range as doubling times observed in parallel T-flask cultures (1.2070.36 days). The carrier cultures, initially seeded with approximately 10 cells per carrier (1 106 cells/100.000 carriers), yielded 400–500 cells per carrier after the 12day expansion phase. 3.2. Pellet redifferentiation in vitro Regardless of expansion method, cell pellets showed no staining for GAG after 7 days in medium 1 (Figs. 3A and D). In insulin-containing medium (2), only carrierexpanded cells demonstrated chondrocyte-like morphology and staining for GAG (Figs. 3B and E). With serum free medium (3) supplemented with growth factors, GAG production in pellets was evident irrespective of expansion method (Figs. 3C and F). However, based on cell shape and GAG staining, pellets of microcarrier-expanded cells demonstrated a higher degree of
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Fig. 3. Proteoglycan staining (safranin-O) of cell pellets from T-flask(A,B,C) and microcarrier- (D,E,F) expanded nasal chondrocytes, cultured for 7 days in medium 1 (A,D), 2 (B,E), and 3 (C,F). Original magnification 40 .
(C) Fig. 2. MTT (A,B) and FDA/PI (C) assay of nasal chondrocytes cultured on CultiSpher G carriers for 3 days (A) and 7 days (B,C). Bar=400 mm.
redifferentiation, whilst pellets of T-flask expanded cells had a more fibrous appearance and stained less intense for GAG.
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3.3. Scaffold redifferentiation in vitro and in vivo After 3 days of culture cells had spread throughout the entire scaffold. Nevertheless, more cells were found within the periphery of the constructs (results not shown). After 14 days of culture localized areas with intense staining for GAG were observed within both constructs (Fig. 4). In addition, a layer of elongated cells was present within the highly cellular outside edge. The DNA assay demonstrated that DNA content gradually increased with time, and no significant
(B) Fig. 4. Proteoglycan staining (safranin-O) of constructs, from either microcarrier- (A) or T-flask- (B) expanded chondrocytes, cultured for 14 days. Original magnification 100 , arrow indicates the polymer scaffold.
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Fig. 6. Proteoglycan staining (safranin-O) of constructs, from either microcarrier- (A) or T-flask- (B) expanded chondrocytes, cultured for 14 days and, subsequently, subcutaneously implanted in nude mice for 14 days. Original magnification 40 , arrow indicates the polymer scaffold.
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Fig. 5. Quantification of DNA (A), glycosaminoglycans (B) and hydroxyproline (C) per tissue engineered construct cultured for either 3 days (3d) or 14 days (14d) in medium 2 or for 14 days in medium 2 and, subsequently, subcutaneously implanted for 14 days in nude mice (14d+14d). (DNA, glycosaminoglycan and hydroxyproline content are expressed as mean7SD and po0:05 was considered significant. =significantly different.)
differences were found between either of the two expansion methods (Fig. 5A). GAG content per scaffold increased from approximately 30 mg at day 3–130 mg at day 14 in vitro. At the in vitro time points, pellets from microcarrier or twodimensionally expanded cells showed no significant difference in total GAG content. After in vivo implantation, GAG content was found significantly higher in ‘14d+14d’ constructs from microcarrier expanded cells (288.5729.2 mg) than in constructs from twodimensionally expanded cells (164.0728.7 mg) (Fig. 5B). These observations correlate with the safranin-O staining at these time points (Fig. 4). At the 14d+14d time point, intense staining for GAG was localized in both constructs (Fig. 6). Nevertheless, more regions demonstrating round cells in lacunae, an indicator of a cartilage-like morphology, were observed within the constructs from microcarrier-expanded cells. Further-
more, more staining for GAG was localized within the outer regions of these constructs (Fig. 6B). Although, total collagen, as measured by hydroxyproline content, did not increase dramatically during 14 days of in vitro culture (Fig. 5C), constructs from Tflask-expanded cells produced slightly more collagen at this time point (not significant). After subcutaneous implantation of the constructs, an increase in collagen content was observed for both conditions. The amount of collagen produced by chondrocytes expanded on microcarriers after 14 days of implantation was slightly higher; however, the difference was not significant.
4. Discussion These results demonstrate that isolated human nasal chondrocytes can be propagated on CultiSpher microcarriers to yield viable cells and comparable expansion rates to T-flask culture. Previous investigations have emphasized that the initial phase of a microcarrier culture is usually the most critical stage, as non-attached or inhomogenously distributed cells have considerable effects on the yield and growth rate obtained [30]. Additionally, it has been reported that mammalian cells, in general, need more time to attach to CultiSpher G microcarriers in comparison to other commonly used microcarriers, such as Cytodex [11]. Therefore, a 24-h
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period of intermittent stirring was applied to assure homogeneous distribution of attached cells throughout the microcarrier culture. Doubling times, obtained in this system (1.0770.14 days) were lower than reported previously for human nasal chondrocytes on microcarriers (74 days) [7]. In vitro redifferentiation of human nasal septum chondrocytes in pellets was performed using three culture media. Medium 1, which has been used by several authors for 3-dimensional bovine chondrocyte culture [31,32], did not yield positive staining for proteoglycans at the time point evaluated (7 days), regardless of the expansion method (Fig. 2). However, pellet cultures in media 2 and 3 demonstrated that redifferentiation (GAG production) was enhanced by microcarrier expansion. These media contain supplements previously shown to stimulate chondrogenesis [2,25]. From these results, an evident question is how culture on microcarriers stimulates the subsequent redifferentiation. The stimulatory effects observed could be related to several specific aspects of the microcarrier culture, including (I) fluid-induced shear, (II) better reproduction of their natural three-dimensional environment and (III) the presence of nutrient gradients, as explained below in greater detail. It has been suggested that the macroporous structure of CultiSpher microcarriers creates a protected environment for the chondrocytes, as the cells within the interior will experience lower shear forces than those encountered with non-porous carriers [11,33]. This protected environment could explain why considerable growth rates were observed. On the other hand, cells proliferating on the outside of the carrier experience shear forces, as do chondrocytes in natural cartilage [34]. Smith and colleagues [34,35] reported stimulatory effects of shear stress applied in vitro on chondrogenesis. More specifically, Frondoza et al. [6] and Bouchet et al. [7] observed enhanced expression of chondrocytespecific proteins during spinner-flask culture with nonporous microcarriers and suggested that articular chondrocytes benefit from a mechanically active tissue culture environment. An alternative explanation for the increase in chondrogenic potential with microcarrier-expanded cells is related to the microenvironment experienced by the cells during the expansion phase. Chondrocytes in vivo are surrounded by their ECM, composed of mainly collagen type II and GAG. Culturing isolated chondrocytes in a three-dimensional gelatin-based carrier more closely mimics the in vivo situation. The porous structure allows attachment in three dimensions and, in addition, the gelatin has, like cartilage, hydrogel-like properties [36,37]. Furthermore, the denatured collagen structure could still contain sequences that can bind to the cell via integrin or other receptors [38]. This could
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contribute to the enhancement of the chondrocytic phenotype during expansion. As a result, increased GAG production, a marker of differentiated chondrocytes, can be observed when cells are, subsequently, seeded in pellets or polymer scaffolds. The enhanced redifferentiation potential of microcarrier-expanded chondrocytes could also be related to nutrient gradients. As a result of multiple cell layers within the macroporous CultiSpher microcarriers, gradients of nutrients, such as oxygen, are likely to develop [39]. In particular, oxygen has been shown to be a controlling agent of developmental processes, such as chondrogenesis [40]. Additionally, several studies have reported that low oxygen tensions stimulate the production of hyaline cartilage specific collagen type II and proteoglycans in vitro [41–44]. Because the distinct morphology and metabolism of chondrocytes depends upon their position in the cartilage tissue, Grimshaw and Mason [45] hypothesized that this may be, at least in part, due to the position of the chondrocyte within the oxygen gradient. Results from pellet cultures after T-flask and microcarrier expansion, support the concept that supplements such as TGF-b2 improve neo-cartilage formation from cells in vitro, as assessed by safranin-O staining. However, such growth factors can be costly and, in some cases, unnecessary. Therefore, in subsequent experiments in which chondrocytes were cultured on PEGT/PBT scaffolds, only medium 2 was used, as it contained a minimum amount of supplements (insulin only), while still showing evidence of cartilage matrix production, as was assessed by GAG formation, after 7 days (Fig. 3E). In the in vitro and following in vivo study, scaffold cultures demonstrated that expanded chondrocytes maintained the ability to differentiate (Figs. 4 and 6). It was also found that microcarrierexpanded chondrocytes produce significantly more GAG after 14 days in vitro followed by 14 days in vivo than two-dimensionally expanded cells. Staining for GAG was not homogeneously distributed, which is likely due to the presence of the non-degraded scaffold. The enhanced production of GAG, a cartilage marker, suggests that microcarrier culture aids expression of the chondrocytic phenotype. Surprisingly, total collagen production after 14 days in vitro culture was higher, although not significant, in constructs from T-flaskexpanded cells. After in vivo culture collagen content increased for both samples, but no significant differences between expansion methods were found. However, the assay used quantifies total collagen produced and cannot distinguish between collagen types. Therefore, quantification of hyaline-specific collagen type II within the constructs was not possible. Previous investigations have shown the potential of macroporous carriers as a delivery system for human adult mesenchymal stem cells for tissue engineering
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applications, such as bone repair [46]. Furthermore, nasal chondrocytes have been shown to be a promising cell source for autologous tissue engineering approaches for the repair of cartilage defects. Increased hyaline cartilage-like tissue formation from nasal chondrocytes over articular chondrocytes, has been reported [2,3]. Our experiments have demonstrated that such carriers are also potent matrices for nasal septum chondrocyte expansion, while simultaneously promoting post-expansion chondrogenesis. Nasal cartilage is subjected to tensile stresses due to the interaction of the collagen network and the swelling pressure generated by proteoglycan–ion interactions [47]. However, unlike articular cartilage, it is non-loadbearing, which may influence how nasal septum chondrocytes behave in a loaded site. Therefore, further research on the behavior of nasal chondrocytes and their extra-cellular matrix in a mechanically loaded environment is essential before they can be used to treat articular defects. With human nasal chondrocytes proliferated on macroporous gelatin-based microcarriers, reparative cells, capable of synthesizing a tissue with a cartilagelike appearance, can be obtained. Although the exact mechanism by which culture on microcarriers enhances the chondrocytic phenotype remains uncertain, combining these cells with a scaffold made from a biocompatible and biodegradable PEGT/PBT material, holds promise for future clinical use in repair of articular cartilage defects.
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