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Culture of Meniscal Chondrocytes on Alginate Hydrogel Matrices
0960–3085/04/$30.00+0.00 # 2004 Institution of Chemical Engineers Trans IChemE, Part C, June 2004 Food and Bioproducts Processing, 82(C2): 126–132
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CULTURE OF MENISCAL CHONDROCYTES ON ALGINATE HYDROGEL MATRICES K. MCCONELL, M. JARMAN-SMITH, K. STEWART and J. B. CHAUDHURI* Centre for Regenerative Medicine, Department of Chemical Engineering, University of Bath, UK
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lginate hydrogels, crosslinked ionically (with calcium ions) and covalently (with 1-ethyl-3-dimethylaminopropyl-N-hydroxysuccinimide carbodiiamide) have been investigated for their suitability as scaffolds for meniscal chondrocyte cell culture. Ovine meniscal chondrocytes were encapsulated at 2.6 0.6 106 cells=ml in 1–2% w=v alginate beads and were also seeded onto the surface of flat alginate hydrogels. Alginate beads were cultured in stirred or static environments on DMEM media with 10% serum. The alginate beads supported cell growth for over 50 days, however, cell numbers and viability in the alginate beads were found to decrease over time, with a greater retention of cell viability in the stirred flask culture. When cultured on flat alginate gels, it was found that a much greater cell count was achieved on gels which had been pre-conditioned with culture media containing chondrocytes as compared with soaking in media alone. Covalently crosslinked gels were found to disintegrate over a 3 day period, and were generally unsuitable for supporting meniscal chondrocyte growth. Keywords: meniscus; cartilage; alginate; chondrocytes; tissue engineering.
INTRODUCTION
to clinicians faced with treating meniscal injuries, usually tears, relate to the complex arrangement of collagen fibres which gives the tissue its unique mechanical properties. Current treatments for cartilage damage include tissue transplantation from human cadavers, synthetic joint replacement, subchondral drilling and transplantation from non-functional parts of the knee. These treatments are not usually successful in the long-term and injuries will commonly reoccur. An alternative method of successful long-term repair and regeneration is therefore sought. Tissue engineering of articular cartilage has been widely studied, resulting in the appearance of new injectable scaffolds for treatment of articular cartilage injury such as Carticel1 (Genzyme, USA). In contrast, considerably less work has been carried out on engineering meniscal cartilage replacements (Sweigart and Athanasiou, 2001). There are two main approaches to tissue engineering the meniscus; both involve cells seeded on a biodegradable scaffold which is followed either by (a) in vivo implantation of the seeded scaffold or (b) bioreactor culture in vitro followed by construct implantation. To enable production of cartilage that is indistinguishable from native tissue, design and development of scaffolds, identification of a cell source and full understanding of the interactions between cells and their surroundings must be achieved. Only a few biomaterials have been investigated for their potential as scaffolds for engineering the meniscus (Sweigart and Athanasiou, 2001). Of these the majority have been synthetic scaffolds, usually variations of PLLA (poly-L-lactic acid) polymer.
The knee joint menisci comprise two semi-lunar wedges of fibrocartilaginous tissue positioned between the femoral condyles and tibial plateaus. Meniscal cartilage comprises primarily type I collagen, proteoglycans, adhesion proteins and water (Sweigart and Athanasiou, 2001). It is sparsely populated with chondrocytes and also contains microvascular endothelial cells. Structurally, the meniscus can be divided into two main zones, the superficial zone containing oval or fusiform cells and the deep zone containing solitary rounded or polygonal cells. The exact cell type comprising meniscal cartilage is still unconfirmed and meniscal cells are sometimes referred to as fibrochondrocytes, as they possess properties of both chondrocytes and fibroblasts (Webber et al., 1985). It has been found that, when cultured ex vivo in monolayer, human meniscus cells show three distinguishable cell types: spread fibroblast-like cells (predominant after 7 days), polygonal cells and small rounded chondrocyte-like cells (Nakata et al., 2001). The meniscus is crucial to joint stability, lubrication and force transmission, and failure to repair damaged tissue can result in joint degeneration. In the adult only the outer one-third of the tissue remains vascularized and injuries which occur outside of the vascularized region do not heal naturally. Other challenges *Correspondence to: Dr J.B. Chaudhuri, Centre for Regenerative Medicine, Department of Chemical Engineering, University of Bath, Bath BA2 7AY, UK. E-mail: [email protected]
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CULTURE OF MENISCAL CHONDROCYTES Sodium alginate, a linear polysaccharide extracted from seaweed, has been used as a scaffold for tissue engineering (Kim and Mooney, 1998), by crosslinking with ionic or covalent bonds to form a hydrogel. Ionic crosslinking with divalent cations may be achieved by contacting an aqueous sodium alginate solution with a solution of divalent cations (i.e. calcium chloride or calcium sulphate), resulting in gelation of the alginate structure. Barium and zinc ions have also been used for crosslinking sodium alginate (Shoichet et al., 1996; Chan et al., 2002). Covalent crosslinking of alginate may be achieved by using 1-ethyl-3dimethylaminopropyl carbodiiamide (EDC)–N-hydroxysuccinimide (NHS) chemistry in the presence of protein. This can improve control of mechanical and swelling properties of alginate hydrogels. A variety of proteins have been used to alter gel mechanical and swelling properties (Lee et al., 2000). Encapsulation in alginate has provided a suitable environment for investigation of controlling collagen and proteoglycan synthesis via use of growth factors (Beekman et al., 1997; Collier and Ghosh, 1995) and for studying the formation of the extracellular matrix (Petit et al., 1996). The encapsulation of articular chondrocytes in alginate has been widely studied, but there are very few reports of work with meniscal chondrocytes. Collier and Ghosh (1995) successfully used alginate beads to encapsulate ovine meniscal chondrocytes and evaluate the effect of transforming growth factor beta. Agarose suspension culture has been used to study meniscal chondrocytes for example, meniscal chondrocytes have been found to maintain their differentiated phenotype when encapsulated in vitro in agarose (Webber, 1990). There appears to have been very little research on the culture of meniscal chondrocytes in vitro with other natural hydrogels such as fibrin, hyaluronate or chitosan. Many of these natural hydrogels and many other synthetic hydrogels have however been used successfully for culture of articular chondrocytes (Suh and Matthew, 2000; Lee and Mooney, 2001). In this paper we show that alginate can be used to encapsulate ovine meniscal chondrocytes, and also that the type of cross-linking affects chondrocyte attachment and proliferation. We have also investigated the effects of static and dynamic culture on meniscal chondrocyte viability. MATERIALS AND METHODS Cell Culture Ovine meniscal chondrocytes (OMC) were isolated using a method adapted from Kuettner et al. (1982). Briefly, menisci were aseptically dissected from ovine hind limbs obtained within 24 h of slaughter (Graystone Ltd, Kingston Upon Hull, UK). Isolated menisci were immersed in phosphate-buffered saline (PBS) containing 0.25% (v=v) gentamycin (Sigma, Poole, UK), excess adipose tissue removed and each meniscus was then cut into small fragments. The tissue fragments were incubated with 10 ml g1 of 0.1% (w=v) pronase-E (VWR International Ltd, Lutterworth, UK) for 3 h at 37 C, with constant mixing. After washing with PBS, tissue fragments were then incubated with 10 ml g1 of 0.2% (w=v) Worthington’s collagenase type 2 (Lorne Laboratories Ltd, Twyford, UK) overnight at 37 C, with constant mixing. Digested tissue
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was filtered through 70 mm cell strainers and the cells pelleted by centrifugation at 1000 rpm for 10 min. Isolated fibrochondrocytes were suspended in Dulbecco’s modified Eagle’s medium (DMEM; Sigma) supplemented with 10% foetal calf serum (FCS; Helena BioSciences Europe, Sunderland, UK), 2 mM L-glutamine (Sigma), 50 IU ml1 penicillin, 50 mg ml1 streptomycin (Sigma) and 1% (v=v) non-essential amino acids (NEAA; Sigma) (termed standard culture media) and seeded at a density of 2 104 cm2. Media was changed after 48 h and the cells fed twice weekly thereafter. Experiments were performed using cells at passage 2. Encapsulation in Alginate Beads Alginate solutions were made by dissolving 2% (w=v) or 4% (w=v) sodium alginate (BDH, Poole, UK) in PBS. This was autoclaved and 1 ml of either solution was added to 1 ml of ovine meniscal chondrocyte cell suspension to give a final seeding density of 2.6 0.6 106 cells ml1 and final alginate concentrations of 1 or 2% (w=v). The cell-alginate suspension was dropped into 40 ml of 0.2 M CaCl2 using a sterile syringe and needle (BD Microlance 3, 20G 1.5). Bead droplets were left for 5 min to solidify and the CaCl2 was then removed by aspiration. The beads were washed twice with DMEM (aspirating after each wash) and submerged in excess standard culture media. The beads were incubated in either static or rod-stirred culture (Techne Ltd, Cambridge, UK). Between three and 10 beads were removed from culture at each time interval and dissolved in 55 mM tri-sodium citrate as detailed by Beekman et al. (1997) to determine cell numbers and viability. Flat Gels Ionic cross-linking A 0.5 ml aliquot of 4% (w=v) sterile sodium alginate solution was pipetted into each well of a 24-well plate. Approximately 1 ml of one of the following solutions was then added to each well to crosslink the alginate and form a gel: CaCl2 (0.05–0.2 M); CaSO4 (0.1–0.2 M); 0.2 M CaCO3; 1:1 (vol:vol) 0.4 M NaCl:0.1 M CaCl2; 2:1 (vol:vol) 0.1 M CaCl2:0.4 M NaCl. The plates were then incubated for up to 62 h at 37 C to allow the alginate to gel. The excess solution was then aspirated and gels washed as follows: Gels were washed twice, 1 h each wash, with an equivolume mix of 0.2 M phosphate buffer (pH 5) and 0.1 M CaCl2. Three washes were carried out in PBS (pH 7), 2–3 min each wash. Finally, three washes were performed using DMEM, with the final wash left on overnight. After removal of DMEM the gels were covered in standard culture media for cell seeding. All flat gels were seeded at a density of approximately 5.3 103 cells cm2. Media was replaced at regular intervals (every 3–4 days). The effect of pre-culturing gels in cell conditioned media on subsequent cell attachment and proliferation was studied as follows: media was recovered from wells containing 1 105 cells. Five days after seeding, two gels of the same composition, were submerged in PBS to remove any unattached cells and dissolved in 55 mM tri-sodium citrate to determine cell numbers and viability (Beekman et al., 1997).
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Covalent crosslinking Varying amounts of EDC (97–200 mg) and NHS (15– 70 mg) were dissolved in 2 ml of sterile distilled water. The EDC=NHS solution was added to 5 ml of 4% (w=w) sodium alginate solution and 1 ml of foetal calf serum. A 1 ml aliquot of this gel solution was placed in each well of a 24-well plate and left in the incubator overnight to gel. The gels were then washed and seeded as described above for the ionically cross-linked flat gels. Analytical techniques All cell and gel images were obtained using a light microscope (Nikon Eclipse TS100 or Nikon DIAphot 300, Hitachi 3CCD with attachment HV-C20). Cell counts and viability A 60 ml volume was collected from cell suspensions obtained by dissolving alginate beads, flat gels or from trypsination of monolayer cultures of OMC. An equivalent volume of trypan blue (0.4% v=v) was added for 5 min at room temperature. Triplicate cell counts were performed for each sample using an improved Neumberg haemocytometer. Histology Alginate beads were embedded in OCT compound (R.A. Lamb, Eastbourne, UK) and snap-frozen in liquid nitrogencooled iso-pentane. Sections of 5 mm were cut using a cryostat, air-dried for 1 h then fixed in ice-cold acetone for 10 min. Sections were stained using haematoxylin and eosin. Surface analysis by scanning electron microscopy (SEM) SEM was used to provide high resolution surface images of alginate beads. Three beads were removed from static culture and frozen in a nitrogen cooled plunge freezing unit. The sample was sputter coated in gold using a current of 40 mA. The temperature was maintained at 148– 163 K for observation of the bead sample surface in the JSM-6210 SEM.
Figure 1. Monolayer culture of ovine meniscal chondrocytes on tissue culture plastic 10 days after seeding. Three different cell morphologies can be observed. This figure is available in colour via www.ingentaselect. com=titles=09603085.htm
CaCl2, 6 days after production; Figure 3). The pores in the alginate structure are visible and are estimated to have a diameter of 0.5–1.0 mm. The white sections are thought to be ice crystals resulting from the freezing process. It can be seen that the pores appear too small to allow passage of the cells from the inside the gel. Stirred and Static Culture of Encapsulated Chondrocytes Figure 4 shows the difference between static growth and stirred culture over a 53 day period. In both cases the cell viability per bead declined with time. An approximately linear decrease in cell viability with time can be observed. The cell viability for the stirred cells remained slightly higher than those in static culture. However, analysis of the two sets of data (student’s t-test) indicate that significant differences between the stirred and static experiments only
RESULTS Alginate Beads Initially, meniscal chondrocytes were cultured as a monolayer on plastic to obtain the required density for encapsulation. The majority of cells in monolayer culture were observed to de-differentiate and assume a spread fibroblast-like appearance (Figure 1). When removed from monolayer culture and encapsulated in alginate beads, cells were observed to have assumed a rounded chondrocyte-like appearance (Figure 2). We studied the effects of the concentration of the CaCl2 cross-linking solution (0.05– 0.20 M) on bead formation, and found that the most stable beads were achieved at 0.20 M (data not shown). The alginate beads containing ovine meniscal chondrocytes were approximately 3.5 mm in diameter. SEM images were taken of the outside of the beads containing meniscal chondrocytes (1% alginate, 5 min cross-linking time in 0.2 M
Figure 2. Ovine meniscal chondrocytes encapsulated within an alginate gel bead. Note the round appearance of the encapsulated cells. This figure is available in colour via www.ingentaselect.com=titles=09603085.htm
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Figure 5. Growth of ovine meniscal chondrocytes in 2% alginate beads (initial cell density 2.6 106 cells=ml), using stirred flask () and static culture (m). (*significance at P ¼ 0.05 level, n ¼ 6.) Figure 3. Scanning electron micrograph of the surface of an alginate bead showing the porous structure.
occur at the later time points. The trends shown in Figure 4 are also seen in measurements of the total cell count with time (data not shown). There is assumed to be no variability between size of beads or the initial number of cells per bead. The only difference between the culture conditions is the mixing provided by the stirrer bar. Figure 5 shows the effect of increasing the alginate concentration on cell viability. In this case there is very little significant difference between the stirred and static cultures. Again it is observed that the cell viability and total cell count (data not shown) decreased with time. Culture of Meniscal Chondrocytes on Flat Gels The effect of different cross-linking reagents and methods on chondrocyte culture was investigated. In this case we seeded chondrocytes onto the surface of flat alginate gels as a simpler experimental system. Alginate gels cross-linked with CaCl2 were observed to support sustained cell attachment, for example, Figure 6 shows chondrocytes maintained on the surface of an alginate gel for 56 days, although cell viability at the end of this period was very low (3%). Note that the majority of cells are rounded but are still attached to
Figure 4. Long-term growth of ovine meniscal chondrocytes in 1% alginate beads (initial cell density 2.6 106 cells=ml), using stirred flask () and static culture (m). (*significance at P ¼ 0.05 level, n ¼ 6.)
the gel surface. Alginate hydrogels ionically cross-linked with calcium sulphate were found to support a smaller degree of cell attachment than for CaCl2 (up to 8 days). Calcium carbonate gels maintained their rigidity but did not support cell attachment (data not shown). Solid crystals of calcium sulphate were observed in gels cross-linked with this chemical. The effect of pre-conditioning the gels by incubating them in the presence of cells was investigated. CaCl2 cross-linked gels were incubated for 2 days before seeding in either the presence of DMEM alone, or DMEM containing cells on tissue culture plastic. In the latter case, the gels were washed to remove any cells that had become attached to the gel, prior to seeding and culture for 5 days. Cell attachment to the pre-conditioned gels (Figure 7A) was significantly improved over the gels that were only soaked in media (Figure 7B). Quantitative analysis of the cell count on the gels showed a 12-fold increase on the preconditioned gel (Figure 8). However, cell attachment after 5 days still only represented a maximum of 17% of the initial cells seeded. No significant difference in cell count was found when the CaCl2 concentration was reduced from 0.2 to 0.1 M (Figure 8). The viability of the cells was measured: the
Figure 6. Meniscal chondrocytes growing on the surface of CaCl2-crosslinked alginate gels after 56 days’ culture. Note that the majority of cells are rounded and attached to gel surface. This figure is available in colour via www.ingentaselect.com=titles=09603085.htm
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Figure 9. Covalently bonded alginate gel 10 days after seeding with meniscal chondrocytes. The gel was prepared with 29 mg NHS and 97 mg EDC and was washed with 0.2 M CaCl2 before seeding. This figure is available in colour via www.ingentaselect.com=titles= 09603085.htm
a solid consistency for more than three days. In one case cells were maintained on the gel for up to 10 days (Figure 9), but experiments were limited due to the instability and liquifaction of the gel after this time. At 10 days the cells had a rounded morphology and were attached to the gel surface, as washing with DMEM did not dislodge them. This gel, however, was the only covalently bonded gels had been washed with CaCl2 after crosslinking, thus there may have been a degree of ionic as well as covalent crosslinking.
Figure 7. CaCl2-cross-linked alginate gels 4 days after seeding with meniscal chondrocytes. Before seeding, the gels had been presoaked for 2 days in DMEM media in the presence (A) and absence (B) of a monolayer of chondrocytes. This figure is available in colour via www.ingenta select.com=titles=09603085.htm
pre-conditioned gels had maintained a cell viability of 83% compared with 44% for gels soaked in media alone. Gels were also covalently cross-linked with EDC=NHS, and for all gel compositions tested the gels did not maintain
Figure 8. Effect of preconditioning flat alginate gels on the growth of meniscal chondrocytes. Gel 1: 0.2 M CaCl2 cross-linker, 2 day DMEM soak; gel 2: 0.1 M CaCl2 cross-linker, 2 day DMEM soak; gel 3: 0.2 M CaCl2 cross-linker, 2 day soak in DMEM and chondrocytes (1 105 cells per gel).
DISCUSSION In this study we observed that monolayer culture on tissue culture plastic and to a lesser extent on alginate gel surfaces were observed to contain three different cell types: polygonal cells chondrocyte-like rounded cells; and fibroblastlike spread cells. Encapsulation in the gels indicated some division and retention of the rounded morphology. We believe that the rounded shape was not an indication of non-viability or apoptosis since subsequent plating on plastic showed good viability. Similar human meniscal chondrocytes sub-populations were observed in monolayer culture on tissue culture plastic (Nakata et al., 2001). The difference in meniscal chondrocyte morphology observed on tissue culture plastic (fibroblastic) and in the alginate beads (rounded chondrocyte-like appearance) have also been observed in vivo (Sweigart and Athanasiou, 2001). Similar observations have been reported for encapsulation of articular chondrocytes (Hauselmann et al., 1994). Our estimation of the pore diameter for calcium crosslinked alginate beads (0.5–1.0 mm) was larger than that reported for alginate cylinders (0.02 mm) by Li et al. (1996) and Shoichet et al. (1996). The difference in values obtained may be explained by the different crosslinking conditions used and primarily by the method of pore size determination. Li et al. (1996) and Shoichet et al. (1996) used diffusion coefficients and a pore size model, in this study the pore size was determined from SEM images.
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CULTURE OF MENISCAL CHONDROCYTES In our studies, the cell count per bead and cell viability were observed to decrease with time. This is in contrast to Masuda et al. (2003) and Beekman et al. (1997), who report an increase in DNA per bead of approximately 2.5 times over 15 days culture for bovine articular chondrocytes encapsulated in alginate beads. In addition, Petit et al. (1996) and Kamada et al. (2002) found an increase in DNA of approximately five times in a similar time period. The decrease in cell number per bead that we observed is thought to be a result of cells (viable or non-viable) either leaving the alginate matrix or the matrix degrading, thus releasing cells. Because of the measured and reported pore diameters in alginate gels it is very unlikely that viable cells have left the matrix. If cell loss is a result of the exterior surface of matrix degrading, then taking bead diameter of 3.5 mm and a volume loss directly proportional to cell loss of 85% (as observed after day 16), the end bead diameter would be 1.9 mm. This could have occurred over 16 days unobserved to the naked eye. One possible explanation is that the alginate matrix is limiting mass transfer of essential nutrients through the bead. This may explain the initial very rapid decrease in cell number. As the cell number reduces, the total demand for the limiting nutrient also reduces until the rate of demand equals rate of mass transfer and cell population stabilizes. Cell loss may therefore be a combined result of the breakdown of non-viable cells (due to mass transfer limitations) and alginate degradation. Increasing the alginate concentration has been reported to reduce the rate of mass transfer through sodium alginate membranes (Grassi et al., 2001). Li et al. (1996) reported that an increase in alginate concentration of only 0.5% reduced the diffusion coefficient for oxygen by 15%. This suggests that the encapsulated cell survival was limited by mass transfer. Beads made with 1% alginate were seen to have a significantly higher cell count than those made with 2% alginate in static culture but not in stirred culture. This suggests that cell death (hence cell loss) may have been affected by an increase in alginate concentration, most likely due to a reduction in nutrient mass transfer rates. Flat alginate gels crosslinked with CaCl2 were the only gels to maintain surface cell attachment for any substantial time, but the cell viability at 57 days was found to be very low. Our quantitative results for cell attachment to CaCl2 crosslinked alginate hydrogels are in agreement with reports by Rowley et al. (1999) and Lee and Mooney (2001) that alginate hydrogels support little cell attachment. Interestingly, we found that if alginate gels were pre-conditioned by soaking in the presence of DMEM and a monolayer of chondrocytes, a 12-fold increase in cell attachment was observed. There was no evidence that the cells in the soaking media had attached to the alginate during the cell media soak. It may have been possible that some chondrocyte-secreted growth factors and signalling molecules were released into the media and then taken up into the gel to subsequently enhance cell attachment. Covalently cross-linked gels did not maintain their rigidity and degraded rapidly under culture conditions within 3 days of production. Different ratios of EDC=NHS=alginate were tried, including those detailed by Bouhadir and Mooney (2002). The failure to obtain a gel which maintained its form may be due to the temperature at which the gels were made (37 C) and unknown effects of the culture media (DMEM) used for washing the gels. Although Lee et al. (2000) have
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produced covalently bonded alginate hydrogels with improved mechanical properties, our experimental method was different in that gels were hydrated in DMEM rather than water, and the temperature of gel storage. The mechanical properties of alginate scaffolds are very important in the regeneration of cartilage because of the mechanical forces that the nascent tissue will be exposed to in order to develop a functional product (Darling and Athanasiou, 2003). The mechanical properties of ionically cross-linked alginate have been shown to vary with alginate concentration, cross-linker type and concentration and crosslinking time (Kuo and Ma, 2001; Shoichet et al., 1996; LeRoux et al., 1999). For example, dissolving the solid sodium alginate in 0.15 M sodium chloride is common practice both in the production of beads and flat gels although it has been found to dramatically reduce mechanical properties (LeRoux et al., 1999). Whilst the majority of previous work has focused on ionic crosslinking of sodium alginate there has been a report that covalently crosslinked alginate increased the gel shear modulus by up to 50% (Lee et al. 2000).
CONCLUSIONS The suitability of meniscal chondrocytes for encapsulation in alginate beads and attachment to alginate gel has been investigated. Cells were observed to have maintained a rounded morphology appearance when encapsulated in alginate. The cell number and viability were found to decrease with time, which may be due to mass transfer limitations of the alginate matrix. The effect of culture method (static and dynamic) was found to affect the cell count and viability. Covalently crosslinked alginate hydrogels were not found to support meniscal cell attachment as the breakdown of the gel structure within 3 days of production prevented cell seeding.
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ACKNOWLEDGEMENT We gratefully acknowledge the financial support of the BBSRC. The manuscript was received 15 July 2003 and accepted for publication after revision 24 March 2004.
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CULTURE OF MENISCAL CHONDROCYTES ON ALGINATE HYDROGEL MATRICES K. MCCONELL, M. JARMAN-SMITH, K. STEWART and J. B. CHAUDHURI* Centre for Regenerative Medicine, Department of Chemical Engineering, University of Bath, UK
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lginate hydrogels, crosslinked ionically (with calcium ions) and covalently (with 1-ethyl-3-dimethylaminopropyl-N-hydroxysuccinimide carbodiiamide) have been investigated for their suitability as scaffolds for meniscal chondrocyte cell culture. Ovine meniscal chondrocytes were encapsulated at 2.6 0.6 106 cells=ml in 1–2% w=v alginate beads and were also seeded onto the surface of flat alginate hydrogels. Alginate beads were cultured in stirred or static environments on DMEM media with 10% serum. The alginate beads supported cell growth for over 50 days, however, cell numbers and viability in the alginate beads were found to decrease over time, with a greater retention of cell viability in the stirred flask culture. When cultured on flat alginate gels, it was found that a much greater cell count was achieved on gels which had been pre-conditioned with culture media containing chondrocytes as compared with soaking in media alone. Covalently crosslinked gels were found to disintegrate over a 3 day period, and were generally unsuitable for supporting meniscal chondrocyte growth. Keywords: meniscus; cartilage; alginate; chondrocytes; tissue engineering.
INTRODUCTION
to clinicians faced with treating meniscal injuries, usually tears, relate to the complex arrangement of collagen fibres which gives the tissue its unique mechanical properties. Current treatments for cartilage damage include tissue transplantation from human cadavers, synthetic joint replacement, subchondral drilling and transplantation from non-functional parts of the knee. These treatments are not usually successful in the long-term and injuries will commonly reoccur. An alternative method of successful long-term repair and regeneration is therefore sought. Tissue engineering of articular cartilage has been widely studied, resulting in the appearance of new injectable scaffolds for treatment of articular cartilage injury such as Carticel1 (Genzyme, USA). In contrast, considerably less work has been carried out on engineering meniscal cartilage replacements (Sweigart and Athanasiou, 2001). There are two main approaches to tissue engineering the meniscus; both involve cells seeded on a biodegradable scaffold which is followed either by (a) in vivo implantation of the seeded scaffold or (b) bioreactor culture in vitro followed by construct implantation. To enable production of cartilage that is indistinguishable from native tissue, design and development of scaffolds, identification of a cell source and full understanding of the interactions between cells and their surroundings must be achieved. Only a few biomaterials have been investigated for their potential as scaffolds for engineering the meniscus (Sweigart and Athanasiou, 2001). Of these the majority have been synthetic scaffolds, usually variations of PLLA (poly-L-lactic acid) polymer.
The knee joint menisci comprise two semi-lunar wedges of fibrocartilaginous tissue positioned between the femoral condyles and tibial plateaus. Meniscal cartilage comprises primarily type I collagen, proteoglycans, adhesion proteins and water (Sweigart and Athanasiou, 2001). It is sparsely populated with chondrocytes and also contains microvascular endothelial cells. Structurally, the meniscus can be divided into two main zones, the superficial zone containing oval or fusiform cells and the deep zone containing solitary rounded or polygonal cells. The exact cell type comprising meniscal cartilage is still unconfirmed and meniscal cells are sometimes referred to as fibrochondrocytes, as they possess properties of both chondrocytes and fibroblasts (Webber et al., 1985). It has been found that, when cultured ex vivo in monolayer, human meniscus cells show three distinguishable cell types: spread fibroblast-like cells (predominant after 7 days), polygonal cells and small rounded chondrocyte-like cells (Nakata et al., 2001). The meniscus is crucial to joint stability, lubrication and force transmission, and failure to repair damaged tissue can result in joint degeneration. In the adult only the outer one-third of the tissue remains vascularized and injuries which occur outside of the vascularized region do not heal naturally. Other challenges *Correspondence to: Dr J.B. Chaudhuri, Centre for Regenerative Medicine, Department of Chemical Engineering, University of Bath, Bath BA2 7AY, UK. E-mail: [email protected]
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CULTURE OF MENISCAL CHONDROCYTES Sodium alginate, a linear polysaccharide extracted from seaweed, has been used as a scaffold for tissue engineering (Kim and Mooney, 1998), by crosslinking with ionic or covalent bonds to form a hydrogel. Ionic crosslinking with divalent cations may be achieved by contacting an aqueous sodium alginate solution with a solution of divalent cations (i.e. calcium chloride or calcium sulphate), resulting in gelation of the alginate structure. Barium and zinc ions have also been used for crosslinking sodium alginate (Shoichet et al., 1996; Chan et al., 2002). Covalent crosslinking of alginate may be achieved by using 1-ethyl-3dimethylaminopropyl carbodiiamide (EDC)–N-hydroxysuccinimide (NHS) chemistry in the presence of protein. This can improve control of mechanical and swelling properties of alginate hydrogels. A variety of proteins have been used to alter gel mechanical and swelling properties (Lee et al., 2000). Encapsulation in alginate has provided a suitable environment for investigation of controlling collagen and proteoglycan synthesis via use of growth factors (Beekman et al., 1997; Collier and Ghosh, 1995) and for studying the formation of the extracellular matrix (Petit et al., 1996). The encapsulation of articular chondrocytes in alginate has been widely studied, but there are very few reports of work with meniscal chondrocytes. Collier and Ghosh (1995) successfully used alginate beads to encapsulate ovine meniscal chondrocytes and evaluate the effect of transforming growth factor beta. Agarose suspension culture has been used to study meniscal chondrocytes for example, meniscal chondrocytes have been found to maintain their differentiated phenotype when encapsulated in vitro in agarose (Webber, 1990). There appears to have been very little research on the culture of meniscal chondrocytes in vitro with other natural hydrogels such as fibrin, hyaluronate or chitosan. Many of these natural hydrogels and many other synthetic hydrogels have however been used successfully for culture of articular chondrocytes (Suh and Matthew, 2000; Lee and Mooney, 2001). In this paper we show that alginate can be used to encapsulate ovine meniscal chondrocytes, and also that the type of cross-linking affects chondrocyte attachment and proliferation. We have also investigated the effects of static and dynamic culture on meniscal chondrocyte viability. MATERIALS AND METHODS Cell Culture Ovine meniscal chondrocytes (OMC) were isolated using a method adapted from Kuettner et al. (1982). Briefly, menisci were aseptically dissected from ovine hind limbs obtained within 24 h of slaughter (Graystone Ltd, Kingston Upon Hull, UK). Isolated menisci were immersed in phosphate-buffered saline (PBS) containing 0.25% (v=v) gentamycin (Sigma, Poole, UK), excess adipose tissue removed and each meniscus was then cut into small fragments. The tissue fragments were incubated with 10 ml g1 of 0.1% (w=v) pronase-E (VWR International Ltd, Lutterworth, UK) for 3 h at 37 C, with constant mixing. After washing with PBS, tissue fragments were then incubated with 10 ml g1 of 0.2% (w=v) Worthington’s collagenase type 2 (Lorne Laboratories Ltd, Twyford, UK) overnight at 37 C, with constant mixing. Digested tissue
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was filtered through 70 mm cell strainers and the cells pelleted by centrifugation at 1000 rpm for 10 min. Isolated fibrochondrocytes were suspended in Dulbecco’s modified Eagle’s medium (DMEM; Sigma) supplemented with 10% foetal calf serum (FCS; Helena BioSciences Europe, Sunderland, UK), 2 mM L-glutamine (Sigma), 50 IU ml1 penicillin, 50 mg ml1 streptomycin (Sigma) and 1% (v=v) non-essential amino acids (NEAA; Sigma) (termed standard culture media) and seeded at a density of 2 104 cm2. Media was changed after 48 h and the cells fed twice weekly thereafter. Experiments were performed using cells at passage 2. Encapsulation in Alginate Beads Alginate solutions were made by dissolving 2% (w=v) or 4% (w=v) sodium alginate (BDH, Poole, UK) in PBS. This was autoclaved and 1 ml of either solution was added to 1 ml of ovine meniscal chondrocyte cell suspension to give a final seeding density of 2.6 0.6 106 cells ml1 and final alginate concentrations of 1 or 2% (w=v). The cell-alginate suspension was dropped into 40 ml of 0.2 M CaCl2 using a sterile syringe and needle (BD Microlance 3, 20G 1.5). Bead droplets were left for 5 min to solidify and the CaCl2 was then removed by aspiration. The beads were washed twice with DMEM (aspirating after each wash) and submerged in excess standard culture media. The beads were incubated in either static or rod-stirred culture (Techne Ltd, Cambridge, UK). Between three and 10 beads were removed from culture at each time interval and dissolved in 55 mM tri-sodium citrate as detailed by Beekman et al. (1997) to determine cell numbers and viability. Flat Gels Ionic cross-linking A 0.5 ml aliquot of 4% (w=v) sterile sodium alginate solution was pipetted into each well of a 24-well plate. Approximately 1 ml of one of the following solutions was then added to each well to crosslink the alginate and form a gel: CaCl2 (0.05–0.2 M); CaSO4 (0.1–0.2 M); 0.2 M CaCO3; 1:1 (vol:vol) 0.4 M NaCl:0.1 M CaCl2; 2:1 (vol:vol) 0.1 M CaCl2:0.4 M NaCl. The plates were then incubated for up to 62 h at 37 C to allow the alginate to gel. The excess solution was then aspirated and gels washed as follows: Gels were washed twice, 1 h each wash, with an equivolume mix of 0.2 M phosphate buffer (pH 5) and 0.1 M CaCl2. Three washes were carried out in PBS (pH 7), 2–3 min each wash. Finally, three washes were performed using DMEM, with the final wash left on overnight. After removal of DMEM the gels were covered in standard culture media for cell seeding. All flat gels were seeded at a density of approximately 5.3 103 cells cm2. Media was replaced at regular intervals (every 3–4 days). The effect of pre-culturing gels in cell conditioned media on subsequent cell attachment and proliferation was studied as follows: media was recovered from wells containing 1 105 cells. Five days after seeding, two gels of the same composition, were submerged in PBS to remove any unattached cells and dissolved in 55 mM tri-sodium citrate to determine cell numbers and viability (Beekman et al., 1997).
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Covalent crosslinking Varying amounts of EDC (97–200 mg) and NHS (15– 70 mg) were dissolved in 2 ml of sterile distilled water. The EDC=NHS solution was added to 5 ml of 4% (w=w) sodium alginate solution and 1 ml of foetal calf serum. A 1 ml aliquot of this gel solution was placed in each well of a 24-well plate and left in the incubator overnight to gel. The gels were then washed and seeded as described above for the ionically cross-linked flat gels. Analytical techniques All cell and gel images were obtained using a light microscope (Nikon Eclipse TS100 or Nikon DIAphot 300, Hitachi 3CCD with attachment HV-C20). Cell counts and viability A 60 ml volume was collected from cell suspensions obtained by dissolving alginate beads, flat gels or from trypsination of monolayer cultures of OMC. An equivalent volume of trypan blue (0.4% v=v) was added for 5 min at room temperature. Triplicate cell counts were performed for each sample using an improved Neumberg haemocytometer. Histology Alginate beads were embedded in OCT compound (R.A. Lamb, Eastbourne, UK) and snap-frozen in liquid nitrogencooled iso-pentane. Sections of 5 mm were cut using a cryostat, air-dried for 1 h then fixed in ice-cold acetone for 10 min. Sections were stained using haematoxylin and eosin. Surface analysis by scanning electron microscopy (SEM) SEM was used to provide high resolution surface images of alginate beads. Three beads were removed from static culture and frozen in a nitrogen cooled plunge freezing unit. The sample was sputter coated in gold using a current of 40 mA. The temperature was maintained at 148– 163 K for observation of the bead sample surface in the JSM-6210 SEM.
Figure 1. Monolayer culture of ovine meniscal chondrocytes on tissue culture plastic 10 days after seeding. Three different cell morphologies can be observed. This figure is available in colour via www.ingentaselect. com=titles=09603085.htm
CaCl2, 6 days after production; Figure 3). The pores in the alginate structure are visible and are estimated to have a diameter of 0.5–1.0 mm. The white sections are thought to be ice crystals resulting from the freezing process. It can be seen that the pores appear too small to allow passage of the cells from the inside the gel. Stirred and Static Culture of Encapsulated Chondrocytes Figure 4 shows the difference between static growth and stirred culture over a 53 day period. In both cases the cell viability per bead declined with time. An approximately linear decrease in cell viability with time can be observed. The cell viability for the stirred cells remained slightly higher than those in static culture. However, analysis of the two sets of data (student’s t-test) indicate that significant differences between the stirred and static experiments only
RESULTS Alginate Beads Initially, meniscal chondrocytes were cultured as a monolayer on plastic to obtain the required density for encapsulation. The majority of cells in monolayer culture were observed to de-differentiate and assume a spread fibroblast-like appearance (Figure 1). When removed from monolayer culture and encapsulated in alginate beads, cells were observed to have assumed a rounded chondrocyte-like appearance (Figure 2). We studied the effects of the concentration of the CaCl2 cross-linking solution (0.05– 0.20 M) on bead formation, and found that the most stable beads were achieved at 0.20 M (data not shown). The alginate beads containing ovine meniscal chondrocytes were approximately 3.5 mm in diameter. SEM images were taken of the outside of the beads containing meniscal chondrocytes (1% alginate, 5 min cross-linking time in 0.2 M
Figure 2. Ovine meniscal chondrocytes encapsulated within an alginate gel bead. Note the round appearance of the encapsulated cells. This figure is available in colour via www.ingentaselect.com=titles=09603085.htm
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Figure 5. Growth of ovine meniscal chondrocytes in 2% alginate beads (initial cell density 2.6 106 cells=ml), using stirred flask () and static culture (m). (*significance at P ¼ 0.05 level, n ¼ 6.) Figure 3. Scanning electron micrograph of the surface of an alginate bead showing the porous structure.
occur at the later time points. The trends shown in Figure 4 are also seen in measurements of the total cell count with time (data not shown). There is assumed to be no variability between size of beads or the initial number of cells per bead. The only difference between the culture conditions is the mixing provided by the stirrer bar. Figure 5 shows the effect of increasing the alginate concentration on cell viability. In this case there is very little significant difference between the stirred and static cultures. Again it is observed that the cell viability and total cell count (data not shown) decreased with time. Culture of Meniscal Chondrocytes on Flat Gels The effect of different cross-linking reagents and methods on chondrocyte culture was investigated. In this case we seeded chondrocytes onto the surface of flat alginate gels as a simpler experimental system. Alginate gels cross-linked with CaCl2 were observed to support sustained cell attachment, for example, Figure 6 shows chondrocytes maintained on the surface of an alginate gel for 56 days, although cell viability at the end of this period was very low (3%). Note that the majority of cells are rounded but are still attached to
Figure 4. Long-term growth of ovine meniscal chondrocytes in 1% alginate beads (initial cell density 2.6 106 cells=ml), using stirred flask () and static culture (m). (*significance at P ¼ 0.05 level, n ¼ 6.)
the gel surface. Alginate hydrogels ionically cross-linked with calcium sulphate were found to support a smaller degree of cell attachment than for CaCl2 (up to 8 days). Calcium carbonate gels maintained their rigidity but did not support cell attachment (data not shown). Solid crystals of calcium sulphate were observed in gels cross-linked with this chemical. The effect of pre-conditioning the gels by incubating them in the presence of cells was investigated. CaCl2 cross-linked gels were incubated for 2 days before seeding in either the presence of DMEM alone, or DMEM containing cells on tissue culture plastic. In the latter case, the gels were washed to remove any cells that had become attached to the gel, prior to seeding and culture for 5 days. Cell attachment to the pre-conditioned gels (Figure 7A) was significantly improved over the gels that were only soaked in media (Figure 7B). Quantitative analysis of the cell count on the gels showed a 12-fold increase on the preconditioned gel (Figure 8). However, cell attachment after 5 days still only represented a maximum of 17% of the initial cells seeded. No significant difference in cell count was found when the CaCl2 concentration was reduced from 0.2 to 0.1 M (Figure 8). The viability of the cells was measured: the
Figure 6. Meniscal chondrocytes growing on the surface of CaCl2-crosslinked alginate gels after 56 days’ culture. Note that the majority of cells are rounded and attached to gel surface. This figure is available in colour via www.ingentaselect.com=titles=09603085.htm
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Figure 9. Covalently bonded alginate gel 10 days after seeding with meniscal chondrocytes. The gel was prepared with 29 mg NHS and 97 mg EDC and was washed with 0.2 M CaCl2 before seeding. This figure is available in colour via www.ingentaselect.com=titles= 09603085.htm
a solid consistency for more than three days. In one case cells were maintained on the gel for up to 10 days (Figure 9), but experiments were limited due to the instability and liquifaction of the gel after this time. At 10 days the cells had a rounded morphology and were attached to the gel surface, as washing with DMEM did not dislodge them. This gel, however, was the only covalently bonded gels had been washed with CaCl2 after crosslinking, thus there may have been a degree of ionic as well as covalent crosslinking.
Figure 7. CaCl2-cross-linked alginate gels 4 days after seeding with meniscal chondrocytes. Before seeding, the gels had been presoaked for 2 days in DMEM media in the presence (A) and absence (B) of a monolayer of chondrocytes. This figure is available in colour via www.ingenta select.com=titles=09603085.htm
pre-conditioned gels had maintained a cell viability of 83% compared with 44% for gels soaked in media alone. Gels were also covalently cross-linked with EDC=NHS, and for all gel compositions tested the gels did not maintain
Figure 8. Effect of preconditioning flat alginate gels on the growth of meniscal chondrocytes. Gel 1: 0.2 M CaCl2 cross-linker, 2 day DMEM soak; gel 2: 0.1 M CaCl2 cross-linker, 2 day DMEM soak; gel 3: 0.2 M CaCl2 cross-linker, 2 day soak in DMEM and chondrocytes (1 105 cells per gel).
DISCUSSION In this study we observed that monolayer culture on tissue culture plastic and to a lesser extent on alginate gel surfaces were observed to contain three different cell types: polygonal cells chondrocyte-like rounded cells; and fibroblastlike spread cells. Encapsulation in the gels indicated some division and retention of the rounded morphology. We believe that the rounded shape was not an indication of non-viability or apoptosis since subsequent plating on plastic showed good viability. Similar human meniscal chondrocytes sub-populations were observed in monolayer culture on tissue culture plastic (Nakata et al., 2001). The difference in meniscal chondrocyte morphology observed on tissue culture plastic (fibroblastic) and in the alginate beads (rounded chondrocyte-like appearance) have also been observed in vivo (Sweigart and Athanasiou, 2001). Similar observations have been reported for encapsulation of articular chondrocytes (Hauselmann et al., 1994). Our estimation of the pore diameter for calcium crosslinked alginate beads (0.5–1.0 mm) was larger than that reported for alginate cylinders (0.02 mm) by Li et al. (1996) and Shoichet et al. (1996). The difference in values obtained may be explained by the different crosslinking conditions used and primarily by the method of pore size determination. Li et al. (1996) and Shoichet et al. (1996) used diffusion coefficients and a pore size model, in this study the pore size was determined from SEM images.
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CULTURE OF MENISCAL CHONDROCYTES In our studies, the cell count per bead and cell viability were observed to decrease with time. This is in contrast to Masuda et al. (2003) and Beekman et al. (1997), who report an increase in DNA per bead of approximately 2.5 times over 15 days culture for bovine articular chondrocytes encapsulated in alginate beads. In addition, Petit et al. (1996) and Kamada et al. (2002) found an increase in DNA of approximately five times in a similar time period. The decrease in cell number per bead that we observed is thought to be a result of cells (viable or non-viable) either leaving the alginate matrix or the matrix degrading, thus releasing cells. Because of the measured and reported pore diameters in alginate gels it is very unlikely that viable cells have left the matrix. If cell loss is a result of the exterior surface of matrix degrading, then taking bead diameter of 3.5 mm and a volume loss directly proportional to cell loss of 85% (as observed after day 16), the end bead diameter would be 1.9 mm. This could have occurred over 16 days unobserved to the naked eye. One possible explanation is that the alginate matrix is limiting mass transfer of essential nutrients through the bead. This may explain the initial very rapid decrease in cell number. As the cell number reduces, the total demand for the limiting nutrient also reduces until the rate of demand equals rate of mass transfer and cell population stabilizes. Cell loss may therefore be a combined result of the breakdown of non-viable cells (due to mass transfer limitations) and alginate degradation. Increasing the alginate concentration has been reported to reduce the rate of mass transfer through sodium alginate membranes (Grassi et al., 2001). Li et al. (1996) reported that an increase in alginate concentration of only 0.5% reduced the diffusion coefficient for oxygen by 15%. This suggests that the encapsulated cell survival was limited by mass transfer. Beads made with 1% alginate were seen to have a significantly higher cell count than those made with 2% alginate in static culture but not in stirred culture. This suggests that cell death (hence cell loss) may have been affected by an increase in alginate concentration, most likely due to a reduction in nutrient mass transfer rates. Flat alginate gels crosslinked with CaCl2 were the only gels to maintain surface cell attachment for any substantial time, but the cell viability at 57 days was found to be very low. Our quantitative results for cell attachment to CaCl2 crosslinked alginate hydrogels are in agreement with reports by Rowley et al. (1999) and Lee and Mooney (2001) that alginate hydrogels support little cell attachment. Interestingly, we found that if alginate gels were pre-conditioned by soaking in the presence of DMEM and a monolayer of chondrocytes, a 12-fold increase in cell attachment was observed. There was no evidence that the cells in the soaking media had attached to the alginate during the cell media soak. It may have been possible that some chondrocyte-secreted growth factors and signalling molecules were released into the media and then taken up into the gel to subsequently enhance cell attachment. Covalently cross-linked gels did not maintain their rigidity and degraded rapidly under culture conditions within 3 days of production. Different ratios of EDC=NHS=alginate were tried, including those detailed by Bouhadir and Mooney (2002). The failure to obtain a gel which maintained its form may be due to the temperature at which the gels were made (37 C) and unknown effects of the culture media (DMEM) used for washing the gels. Although Lee et al. (2000) have
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produced covalently bonded alginate hydrogels with improved mechanical properties, our experimental method was different in that gels were hydrated in DMEM rather than water, and the temperature of gel storage. The mechanical properties of alginate scaffolds are very important in the regeneration of cartilage because of the mechanical forces that the nascent tissue will be exposed to in order to develop a functional product (Darling and Athanasiou, 2003). The mechanical properties of ionically cross-linked alginate have been shown to vary with alginate concentration, cross-linker type and concentration and crosslinking time (Kuo and Ma, 2001; Shoichet et al., 1996; LeRoux et al., 1999). For example, dissolving the solid sodium alginate in 0.15 M sodium chloride is common practice both in the production of beads and flat gels although it has been found to dramatically reduce mechanical properties (LeRoux et al., 1999). Whilst the majority of previous work has focused on ionic crosslinking of sodium alginate there has been a report that covalently crosslinked alginate increased the gel shear modulus by up to 50% (Lee et al. 2000).
CONCLUSIONS The suitability of meniscal chondrocytes for encapsulation in alginate beads and attachment to alginate gel has been investigated. Cells were observed to have maintained a rounded morphology appearance when encapsulated in alginate. The cell number and viability were found to decrease with time, which may be due to mass transfer limitations of the alginate matrix. The effect of culture method (static and dynamic) was found to affect the cell count and viability. Covalently crosslinked alginate hydrogels were not found to support meniscal cell attachment as the breakdown of the gel structure within 3 days of production prevented cell seeding.
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ACKNOWLEDGEMENT We gratefully acknowledge the financial support of the BBSRC. The manuscript was received 15 July 2003 and accepted for publication after revision 24 March 2004.
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