ANNALS OF ANATOMY
Effects of hyaluronic acid on the morphology and proliferation of human chondrocytes in primary cell culture E.-M. Elders, P. Behrens*, L. Wiinsch**, W. Kiihnel and M. Russlies* Institut ftir Anatomie, *Klinik fiir Orthop/idie und **Klinik fiir Kinderchirurgie der Medizinischen Universit~it Liibeck, Ratzeburger Allee 160, D-23538 Liibeck, Germany
Summary. Hyaline articular cartilage is a specialised connective tissue with weight bearing and adsorbing functions. Injury or loss of which often leads to impaired joint function and severe pain. Since the self-renewing abilities of hyaline articular cartilage are limited, there is major interest in the development of bioengineered cartilaginous implants. A cell-matrix-biocomposite composed of a collagen I/III scaffold seeded with autologous chondrocytes is currently being used in clinical trials; however, in order to optimise culture conditions, we cultured human condrocytes and seeded them on type I/III collagen membranes and on Thermanox plastic coverslips with media containing 0 to 500 gg/ml Hyaluronic Acid. After 4 days, the cells were either fixed or BrdU incorporation procedures begun. H E staining clearly demonstrated that cells grown in H A form three dimensional clusters and produce secretory vesicles as opposed to the monolayer control cells with noticeably fewer secretory vesicles. BrdU incorporation revealed a noticeable increase in cell proliferation in cells grown in 100 pg/ml; however, no comparable increase in 500 gg/ml but rather a slight depression in proliferation. Immunohistochemistry for collagen If and aggrecan revealed an obvious increase in deposition of these two substances with increased H A administration as compared to the control; however, again, the higher concentration of HA, 500 gg/ml, did not result in a further increase in production. These results suggest that H A at 100 gg/ml not only influences chondrocytes to differentiate and produce more Collagen II and aggrecan, but also increases proliferation. We, therefore, propose that the addition of H A at low to middle dosages in condrocyte culturing might help improve condrocyte redifferentation and thus, the bioengineered cartilage. Correspondence to: E.-M. Ehlers E-mail: [email protected]
Ann Anat (2000) 183: 13-I 7 © Urban & Fischer Verlag http:llwww,urbanfischer,deljournalslannanat
Key words: Articular cartilage - Tissue engineering Chondrocyte culture - Collagen sponge - Hyaluronic acid-morphology
Introduction Hyaline articular cartilage is a specialized connective tissue with weight bearing and adsorbing functions. Injury or loss of articular cartilage often leads to impaired joint function and severe pain. The self-renewing abilities of hyaline articular cartilage are limited and depend upon various factors such as depth and extension of the lesions, the localization of the defect and the age of the patient. Thus, there exists major orthopedic interest in the development of bio-engineered cartilaginous implants for the treatment of localized cartilage defects. Therefore, morphological and biochemical features of this tissue are being investigated in order to assemble most cartilagelike grafts for transplantation. Some features seem to be of substantial importance for the correct function of hyaline articular cartilage: 1) a high content of collagen II compared to fibrous tissue or fibrocartilage, which contain rather collagen I and III, 2) a high content of acidic, sulfated proteoglycans and 3) a special arrangement of cells in isogenic groups with distinct pericellular matrix distribution. The aim of recent investigations is to produce a morphologically and functionally useful cartilage replacement. Different investigators have shown promising results when seeding chondrocytes on various scaffolds in order to induce redifferentiation of these formerly in culture dishes dedifferentiated cells (Frenkel et al. 1997; Liu et al. 1998). Other investigations focus on the redifferentiation of cultured chondrocytes by means of adding cartilage specific growth factors (Galera et al. 1992; Kato et al. 1987; Okazaki et al. 1996). 0940-9602/01/183/1-13 $15.00/0
In p r e c e e d i n g investigations, we have studied the characteristics of a cell-matrix-biocomposite, c o m p o s e d of a collagen I/III-scaffold ( C h o n d r o - G i d e TM, Geistlich Biomaterials, Wolhusen, Switzerland) s e e d e d with autologous c h o n d r o c y t e s (Behrens et al. 1999; Ehlers et al. 1999), which has now a l r e a d y being i n t r o d u c e d into clinical trials. Nevertheless, we are still trying to optimize culture conditions and differentiation with several experiments. The p r e s e n t study focusses on the m o r p h o l o g i c a l effects of the i n t r o d u c t i o n of hyaluronic acid ( H A ) into the cul-
ture m e d i u m of chondrocytes cultured on plastic surfaces and on C h o n d r o - G i d e TM.
Materials and methods Cartilage from human patients was collected under informed consent during total hip or knee replacement, diced into 1 to 2 mm 3 pieces and placed in a spinner flask containing 0.1% collagenase (Sigma C9891) and 0.5% hyaluronidase (Sigma H3506)
Figs. 1 to 3: Light microscopy. Chondrocytes grown on plastic surfaces with different H A concentrations. Fig. 1: without HA, the cells grow mostly as a monolayer and show only a minor tendency to organize as clusters (arrows). Fig. 2: chondrocytes grown on plastic under the influence of 100 gg/ml HA. Note the formation of cell clusters (arrows). Fig. 3- Chondrocytes grown on plastic under the influence of 500 pg/ml HA. The three dimensional network becomes even larger (arrows). HE-staining, x 140. Figs. 4 to 6: Scanning electron microscopy of chondrocytes grown on Chondro-GideTM at different H A concentrations. Note the increase in microvilli and secretory vesicles from 0 pg/ml H A (Fig. 4) to 100 ~tg/ml H A (Fig. 5) (arrows). Adding 500 gg/ml H A to the culture medium (Fig. 6) seems to lead to even more secretory vesicles and microvilli. Magnif. x 1400. 14
for 8 h at 37 °C. The cell suspension was then fltered through a nylon mesh filter to remove remnants of cartilage matrix, centrifuged and resuspended in a culture medium containing Ham's F-12 (Biochrom KG FG0815), 12% fetal calf serum (Biochrom KGS0115), 50gl/ml penicillin/streptomycin (Biochrom KGA2213), 50 gl/ml glutamine (Biochrom KGK0282), and 50 }al/ml non-essential amino acids (Biochrom KG K0363). Cells were cultured to confluency in 80 cm2 culture flasks at 37 °C in an 5% CO2-containing water-saturated atmosphere during 4 to 6 weeks. Cells were then trypsinized, counted and seeded on the type I/III collagen membranes (ChondrogideTM,Geistlich Biomaterials) or on Thermanox plastic coverslips at a density of 106 cells/cm2 into 24 well plates with media containing 0 to 500 gg/ ml hyaluronic acid for 4 days. For light microscopy, specimens were fixed with Bouin's fixative or methanol/acetone 1 : f, dehydrated through a graded ethanol series and stained with Mayer's hemalaun-erythrosin staining. Immunofluorescence was carried out by incubating specimens with 10% normal goat serum in PBS, followed by an overnight incubation of primary antibodies against collagen II and aggrecan (Rabbit anti collagen II, Rockland, PA, USA, 1 : 50, and mouse anti aggrecan, DPC Biermann, Bad Nauheim, Germany, 1:50) diluted in PBS. Specimens were then washed twice with PBS and incubated with FITC-conjugated secondary antibodies (Sigma) before being mounted in Glycergel (Dako) containing 50 mg/ml DABCO (Dako) as an anfifade. Bromodeoxyuridine (BrdU, Becton Dickinson) is a uridine derivative which is incorporated into the DNA during the S phase of the cell cycle, replacing thymidine. Anti-BrdU subsequently added attaches to form an antigen : antibody complex, thus marking cells synthesizing DNA. The procedure was carried out according to the supplier's instructions and specimens were finally counterstained with Mayer's hemalaun. Cells were seeded in triplicates from each of which 1000 cells were counted. For transmission electron microscopy, specimens were fixed after 3, 6, 9 and 12 days with 2.5% glutaraldehyde in a 0.06 M sodium cacodylate buffer (pH 7.35) for 48 to 72 hours at 4 °C. The samples were then rinsed in 0.2 M sodium cacodylate buffer and postfixed with 1% osmium tetroxide in the same buffer, pH 7.35, at room temperature. After being rinsed in 2.4% sodium chloride solution, the samples were washed in 0.2 M sodium acetate buffer (pH 5.0) and block stained with 1% uranyl acetate in 0.2 M sodium acetate buffer (pH 5.0) in the dark for 30 rain. at room temperature. Routine procedures were then followed for dehydration in alcohol and embedding in araldite. Ultrathin sections (60 nm) were stained with lead citrate (Reynolds 1963) and examined in a Philips 400 electron microscope. For scanning electron microscopy, cells were rinsed with warm PBS, fixed with 2% glutaraldehyde and 0.6% parafomaldehyde in a 0.06 M sodium cacodylate buffer, pH 7.35, for 24 hours at 4 °C, dehydrated in graded series of acetone, dried in a critical-point dryer and sputter-coated with gold-paladium and examined in a Philips SEM505 scanning electron microscope operated at 30 kV. The findings were documented on APX100 films (Agfa) for electron microscopy and on Kodak Elite 400 for immunofluorescence.
exhibit a spinocellular, fibroblast-like p h e n o t y p e and grow in a m o n o l a y e r . W i t h o u t adding H A our chondrocytes grow in exactly this m a n n e r (Fig. 1), only rarely showing f o r m a t i o n of cell groups or clusters. A l r e a d y at a dosage of 100 gg/ml (Fig. 2), H A i n d u c e d the f o r m a t i o n of clusters growing t h r e e dimensionally out of the m o n o layer which could be easily d e t e c t e d in the H E stainings. A t a c o n c e n t r a t i o n of 500 gg/ml H A (Fig. 3) most of the cells were growing as clusters in a n e t w o r k with only very few cells growing outside of this n e t w o r k as a monolayer. Light microscopically, t h e r e s e e m e d to be m o r e matrix d e p o s i t i o n b e t w e e n the cells u n d e r the influence of H A . Therefore, we e x a m i n e d the effects of H A on ehondrocytes by means of scanning e l e c t r o n microscopy and found in b o t h cases, for cells grown on plastic and also for cells grown on C h o n d r o - G i d e TM,an increase of secretory vesicles on the surface of the cells. Figures 4 to 6 dem o n s t r a t e these findings. While cells growing u n d e r s t a n d a r d conditions possess only few microvilli and sparsely distributed secretory vesicles on their surface, a treatm e n t with 100 gg/ml H A led to a visible increase of secretory vesicles and microvilli (Fig. 5). A t a concentration of 500 gg/ml H A (Fig. 6), changes in cellular aspect were even m o r e evident. These findings were further d o c u m e n t e d with transmission electron m i c r o s c o p y where an increase in secretory vesicles was also evident (Figs. 7, 8). A s we w a n t e d to k n o w of which n a t u r e these secretory products were, we tested specimens i m m u n o h i s t o chemically for collagen II and aggrecan. F o r b o t h cartilage specific extracellular m a t r i x components, collagen II and aggrecan, t h e r e was an obvious increase of
Results It is k n o w n that H A is the integral core protein, necessary for p r o t e o g l y c a n assembly, W e w a n t e d t h e r e f o r e to know, w h e t h e r adding this i m p o r t a n t substrate to our culture media, could optimize cell growth and/or differentiation. C h o n d r o c y t e s grown on plastic surfaces n o r m a l l y
Figs. 7 and 8 show transmission electron microscopy of chondrocytes treated with 100 ~tg/ml HA; note the high amount of secretory vesicles on the surface of the cell (arrow). x 920 and x 3100. 15
immunoreactivity with increased H A administration, which is shown in figures 9 to 14. However, there does not seem to be a proportional increase above a dosage of 100 gg/ml HA. Collagen II and aggrecan immunoreactivity is much stronger at 100 pg/ml H A (Figs. 10, 13) than under controll conditions (Figs. 9, 12), but further increase of H A concentration up to 500 gg/ml (Figs. 11, 14) does not further increase the collagen II and aggrecan production. A marked increase of differentiation is often accompanied by a decrease in proliferative activity of
cells which can be, among other methods, evaluated by measuring the S phase fraction of a cell population. Therefore, we investigated the BrdU incorporation into DNA immunohistochemically after treatment with different H A concentrations. Interestingly enough, the S phase fraction of the cultured chondrocytes is markedly elevated after administration of 100gg/ml HA, whereas higher levels of H A in the culture media did not lead to a further increase of DNA synthesizing cells, but seemed to somehow suppress entering of cells into S phase (Fig. 15).
Figs. 9 to 11: Immunocytochemistry for Aggrecan. (Fig. 9:0 ~tg/ml; Fig. 10:100 pg/ml; Fig, 11:500 ~tg/ml HA). Note the increase of immunoreactivity in Figs. 10 and 11 compared to Fig. 9, x 140. Figs. 12-14: Immunocytochemistry for Collagen II. (Fig. 12:0 gg/ml; Fig. 13:100 gg/ml HA; Fig. 14:500 gg/ml). Note the increase of immunoreactivity in Figs. 13 and 14 compared to Fig. 12. x360. 16
Influence of hyaluronic acid in different concentrations on BrdU-incorporation in cultured chondrocytes
stimulatory effect of low dosages of H A on the proliferation of chondrocytes, measured by BrdU incorporation into the D N A during S-phase, whereas higher concentrations of H A rather led to a decrease in S-phase fraction and thus proliferation. These results suggest a double action of H A on chondrocytes: They not only show a greater tendency to differentiate, but also a higher rate of proliferation. This double action is very interesting in thus far that differentiation-inducing stimuli are often said to lead to a depression of cell proliferation. Therefore, we propose that adding H A to chondrocyte culture media might help to improve chondrocyte redifferentiation and thus the bio-engineered cartilage.
Fig. 15 shows data of the BrdU incorporation of HA-treated chondrocytes versus controls. Note the significant increase of cells in S phase after treatment with 100 gg/ml HA versus 0 gg/ ml HA. Interestingly enough, administration of 500 pg/ml does not alter or rather suppresses entering of cells into S phase.
References Behrens E Ehlers EM, K6chermann KU, Rohwedel J, Russlies M, P16tz W (1999) Neues Therapieverfahren far lokalisierte Knorpeldefekte. MMW-Fortschr Med 141:793-795 Blaschke RJ, Howlett AR, Desprez PY, Petersen OW, Bissell MJ (1994) Cell differentiation by extracellular matrix components. Methods Enzymol 245:535-557 Bodo M, Pezzetti F, Baroni T, Carinci F, Arena N, Nicoletti I, Becchetti E (1993) Hyaluronic acid modulates growth, morphology and cytoskeleton in embryonic chick skin fibroblasts. Int J Dev Biol 37:349-352 Damsky CH, Werb Z (1992) Signal transduction by integrin receptors for extracellular matrix: cooperative processing of extracellular information. Curt Opin Cell Biol 4:772 Ehlers EM, Ful3 M, Rohwedel J, Russlies M, K(ihnel W, Behrens P (1999) Development of a biocomposite to fill out articular cartilage lesions. Light, scanning and transmission electron microscopy of sheep chondrocytes cultured on a collagen I/III sponge. Ann Anat 181:513-518 Frenkel SR, Toolan B, Menche D, Pitman MI, Pachence JM (1997) Chondroeyte transplantation using a collagen bilayer matrix for cartilage repair. J Bone Joint Surg (Br) 79:831-836 Fug M, Ehlers EM, Russlies M, Rohwedel J, Behrens P (2000) Characteristics of human chondrocytes, osteoblasts and fibroblasts seeded on a type I/III collagen sponge under different culture conditions. A light, scanning and transmission electron microscopic study. Ann Anat 182:1-8 Galera R Redini E Vivien D, Bonaventure L Penfornis H, Loyau G, Pujol JP (1992) Effect of transforming growth factor /~1 (TGF-/~I) on matrix synthesis by monolayer cultures of rabbit articular chondrocytes during the dedifferentiation process. Exp Cell Res 200:379-392 Kato Y, Iwamoto M, Koike T (1987) Fibroblast growth factor stimulates colony formation of differentiated chondrocytes in soft agar. J Cell Physiol 133:491-498 Liu H, Lee YW, Dean MF (1998) Re-expression of differentiated proteoglycan phenotype by dedifferentiated chondrocytes during culture in alginate beads. Biochim Biophys Acta 1425: 505-515 Okazaki R, Sakai A, Makamura T, Kunugita N, Norimura T, Suzuki K (1996) Effects of transforming growth factor fl and basic fibroblast growth factor on articular chondrocytes obtained from immobilized rabbit knees. Ann Rheum Dis 55:181-186 Reynolds ES (1963) The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. J Cell Biol 17:208-212
This shows a dose dependent relationship with small amounts of added H A stimulating chondrocyte proliferation whereas high amounts of H A lead to a slight decrease of chondrocyte proliferation.
Discussion Since the number of patients undergoing surgery because of arthrotic degeneration of hyaline articular cartilage is rising, there is the need for cartilage defect repair before it comes to degenarative changes such as arthrosis. Bioengineered cartilage is the focus of interest for many investigators. Previously we could show the development of a bioengineered chondrocyte-matrix-biocomposite (Ehlers et al. 1999; Ful3 et al. 2000), which has now been introduced into a clinical trial for cartilage defect repair. Nevertheless, there might be a way to possibly further improve the outcome of this biocomposite. We tried therefore to make the chondrocytes "feel more at home" by enriching the culture media with hyaluronic acid, an important component of the hyaline articular extracellular matrix. It is known, that H A modulates growth and morphology of embryonic chick skin fibroblasts in so far in that it inhibits cell multiplication and modifies cell adhesion to the substrate (Bodo et al. 1993). We thought however, that H A might have a positive effect on the redifferentiation of chondrocytes grown on plastic and on a collagen I/III-sponge. Indeed, H A led to an increase of E C M secretion by the ehondrocytes, which resulted in an increase of collagen II and aggrecan deposition. For this reason, we suggest that H A might stimulate differentiation of chondrocytes, probably by binding to surface receptors. For tumor cells and m a m m a r y gland epithelial cells, the influence of E C M components on cellular differentiation is well documented (Review: Blaschke et al. 1994) and even mechanistical studies on E C M components and their receptors have been performed (Damsky and Werb 1992). Interestingly enough, we could show a
Accepted May 29, 2000 17