- Email: [email protected]
Integration of engineered cartilage
Journal of Orthopaedic Research
ELSEVIER
Journal of Orthopaedic Research 19 (2001) 1089-1097 www.elsevier.com/locate/orthres
Integration of engineered cartilage S. Treppo B. Obradovic b, I. Martin a, R.F. Padera G. Vunjak-Novakovic a,* a
a,
L.E. Freed
a,
Diuision of Health Sciences and Techtiology, Massachusetts Institute of Technology, E2.5 Room 330, 45 Carleton Street, Cirmbridge, M A 02139, USA Depurtment of Chemiral Engineering, Tufts Uniiiersity, Medford, M A 02155, USA Harvard Medical School. Boston, M A 02115, U S A
Received 3 April 2000; accepted 31 January 2001
Abstract The structure and function of cartilaginous constructs, engineered in vitro using bovine articular chondrocytes, biodegradable scaffolds and bioreactors, can be modulated by the conditions and duration of tissue cultivation. We hypothesized that the integrative properties of engineered cartilage depend on developmental stage of the construct and the extracellular matrix content of adjacent cartilage, and that some aspects of integration can be studied under controlled in vitro conditions. Disc-shaped constructs (cultured for 5 i 1 days or 5 1 weeks) or explants (untreated or trypsin treated cartilage) were sutured into ring-shaped explants (untreated or trypsin treated cartilage) to form composites that were cultured for an additional 1-8 weeks in bioreactors and evaluated biochemically, histologically and mechanically (compressive stiffness of the central disk, adhesive strength of the integration interface). Immature constructs had poorer mechanical properties but integrated better than either more mature constructs or cartilage explants. Integration of immature constructs involved cell proliferation and the progressive formation of cartilaginous tissue, in contrast to the integration of more mature constructs or native cartilage which involved only the secretion of extracellular matrix components. Integration patterns correlated with the adhesive strength of the disc-ring interface, which was markedly higher for immature constructs than for either more mature constructs or cartilage explants. Trypsin treatment of the adjacent cartilage further enhanced the integration of immature constructs. 0 2001 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved.
*
Introduction It has long been known that mature articular cartilage has a limited capacity for regeneration after degeneration or injury [23]. Methods used clinically to enhance natural repair of articular surfaces include modifications of the damaged surface (chondral shaving with debridement, abrasion arthroplasty, subchondral drilling or microfracturing of the subchondral plate) and transplantation of periosteal, perichondral or osteochondral autografts [5]. In most cases, these methods temporarily relieve symptoms and induce an initial hyaline-like repair, but the repair tissue eventually degenerates to fibrocartilage and the symptoms return. None of the currently available methods can predictably restore a durable articular surface [3].
*Corresponding author. Tel.: +I-617-452-2593; fax: +I-617-2588827. E-mail address: [email protected] (G. Vunjak-Novakovic).
Novel approaches to cartilage repair include the use of autologous or allogenic chondrocytes or chondroprogenitor cells injected at the defect site, alone or in conjunction with collagen gels, fibrin, ceramic, and degradable or non-degradable synthetic polymers [22]. Although these approaches are promising, long-term maintenance of regenerated hyaline tissue under conditions of normal joint loading was variable and inconsistent [5]. Systematic studies are needed to determine the specific factors (e.g., cells, biomaterials, enzymes, mechanical loading) governing the integration of transplanted cells or tissues into the site of a chondral or osteochondral defect. Although cell-based grafts have been studied in animal models with respect to the choice of carrier and the methods of graft fixation [22,24,29,33,34], the criteria for graft selection and the mechanisms of graft-host integration are not yet well understood. The variability of the in vivo environment further complicates evaluation of the results. Cartilage tissue engineering has been motivated by the need to replace lost or damaged tissue with an
0736-0266/01/$ - see front matter 0 2001 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved. PII: S 0 7 3 6 - 0 2 6 6 ( 0 1 ) 00 0 3 0 - 4
1090
B. Obradovic et al. I Journal of Orthopaedic Research 19 (2001) 1089-1097
already structurally and mechanically functional implant that can be created in vitro using chondrocytes or chondroprogenitor cells in conjunction with biomaterials [4,12-151. We have previously demonstrated that cartilaginous constructs engineered using articular chondrocytes, biodegradable polymer scaffolds and bioreactors had structure (biochemical composition, histomorphology) and function (mechanical behavior, biosynthesis rates) that depended on the conditions and duration of bioreactor cultivation [32]. After 6 weeks in culture, constructs were cartilaginous throughout their volume [l 11, and had wet weight fractions of glycosaminoglycan (GAG) and collagen and equilibrium moduli of 78%, 43% and 25% of the respective values measured for freshly explanted cartilage 1321. After prolonged (7 months) cultivation, wet weight fractions of GAG and equilibrium moduli of engineered constructs were comparable or exceeded values measured for cartilage [lo]. In the present work, the integration of engineered constructs with native cartilage was studied in disdring composites cultured in bioreactors for up to 8 weeks. We hypothesized that the integrative properties of engineered constructs can be modulated by the duration of bioreactor cultivation (which determines cell biosynthetic activity and construct structure, composition, and mechanical behavior) and by trypsin treatment of the adjacent cartilage (which determines tissue concentration of GAG).
Materials and methods Cartilaginous constructs
Cartilage was engineered as previously described [10,32] using articular chondrocytes, biodegradable polyglycolic acid (PGA) scaffolds and bioreactors. Full thickness articular cartilage was harvested from the femoropatellar grooves (FPG) of 2-3 week old bovine calves within 8 h of slaughter. Chondrocytes were isolated using type I1 collagenase (Worthington, Freehold, NJ) and resuspended in culture medium (Dulbecco's Modified Eagle Medium (DMEM) containing 4.5 gA glucose and supplemented with 10% fetal bovine serum, 10 mM N-2hydroxyethylpiperazine "-2-ethane sulfonic acid (HEPES), 50 Ulml penicillin, 50 &ml streptomycin, 0.1 mM non-essential amino acids, 0.4 mM proline, 50 pg/ml ascorbic acid, 0.5 pg/ml fungizone) [4]. Scaffolds were fibrous disks, 5 mm in diameter x 2 mm thick, made of biodegradable PGA by Albany International (Mansfield, MA) as a highly porous mesh with a bulk density of 65 mg/cm3 and a void volume of 97% [8]. Scaffolds were dynamically seeded with freshly isolated chondrocytes as previously described [31], using 12 scaffolds per flask in 120 cm3 of cell suspension containing 5 x los cellslcm3 and stirred at 50 rpm. Gas exchange was provided by surface aeration (10% C 0 2 in humidified air). After 5 i 1 days of seeding, constructs were either harvested and used for composite preparation ( 5 day constructs) or cultured in bioreactors for an additional 5 f 1 weeks (5 week constructs). Twelve constructs per bioreactor were cultured in 110 cm3 volume annular space, as previously described [9,15]. Each bioreactor was rotated as a solid body around the horizontal axis; the rotation rate was adjusted to keep constructs freely settling in rotating flow, and gradually increased over time to accommodate the increase in construct size and weight. Gas exchange was provided by diffusion
through the inner cylinder. Culture medium was completely replaced after seeding, and then by 50% vlv every other day for the duration of cultivation. Cartilage explants
Natural cartilage plugs (10 mm in diameter) were harvested from cartilage cores obtained from the medial and lateral ridges adjacent to the FPG using a dermal punch (Miltex Instruments, Lake Success, N Y ) . The thickness of these cores always exceeded 4.5 mm. Discs (10 mm diameter x 2 mm thick) were obtained from the middle portion of the cores after removing 1 nim thick slices from both the articular surface and the regon adjacent to the subchondral bone, and rinsed with phosphate buffered saline supplemented with 100 U/cm3 penicillin and 50 pg/cm3 streptomycin. Dermal punches were then used to core the 10 mm diameter discs into final explant rings (outer and inner diameters 10 and 5 mm, respectively, 2 mm thick, in all groups) and explant discs (5 mm diameter x 2 mm thick, in all groups). In some groups, explant rings and discs were treated by trypsin (cell culture quality, type I, from Gibco, 1% in PBS at 37"C), by evenly exposing all surfaces to the enzyme solution. The duration of incubation was determined empirically and set to either 10 or 20 min, to reproducibly remove GAG from a -0.6 or 1.2 mm thick peripheral zone, as assessed from safranin-0 stained histological sections. Incubation time was set to 10 min when both rings and discs were treated, and 20 min when only rings were treated, such that the total thickness of the treated zone at the tissue interface was -1.2 mm. Composites
Discs ( 5 mm in diameter x 2 mm thick in all groups) were press-fit into the rings (1015 mm in diameter x 2 mm thick in all groups) and sutured together using #5-0 silk surgical suture and cutting FS-2 needle, with two stitches positioned at a 90" angle from each other. The disc and ring were connected at points that were approximately 1 mm away from the integration interface (Fig. 1). Six groups of composites were established using four different component tissues: constructs cultured for 5 days or 5 weeks, untreated or treated explants, as follows. Group 1-3 composites had central discs that were 5 day constructs, 5 week constructs or untreated explants, respectively, and outer rings that were intact explant rings. Group 4 6 composites had central discs that were 5 day constructs, 5 week constructs or treated explants (0.6 mm thick peripheral zone), respectively, and outer rings that were treated explants (1.2 mm thick peripheral zone in groups 4 and 5; 0.6 mm thick peripheral zone in group 6 ) . Composites were cultured for 1-8 weeks in rotating bioreactors (n = 12 composites from one experimental group per bioreactor) as described above for engineered constructs. Final rotation rates were as high as 45 rpm. The experiments involved a total of 87 engineered constructs, 151 explants and 95 composites (12-24 per experimental group), using articular cartilage obtained from 21 bovine knee joints. For each experimental group, composites were sampled at timed intervals and analyzed biochemically (discs and rings from n = 3 composites per data point), histologically (n = 2-3 composites per data point), and mechanically (discs from n = 3 4 composites per data point to measure equilibrium modulus; n = 4 composites per data point to measure adhesive strength), as follows. Biochemical analyses
Composites were sampled at the time of preparation, after 4 and 8 weeks of cultivation, and separated into discs and rings along the integration interface using a surgical blade. The disc and ring could be distinguished in all cases by careful macroscopic inspection of the integration interface. Samples were frozen, lyophilized and digested with 0.125 pg/ml papain (Sigma, St. Louis, MO) in 100 mM phosphate buffer (PBS) with 10 mM EDTA and 10 mM cysteine for 15 h at 60°C [27], using 1 ml enzyme solution per 4-40 mg dry weight of the sample. The number of chondrocytes was obtained from the measured amount of DNA, using Hoechst 33258 dye and spectrofluorometer (PTI, South Brunswick, NJ) [21], based on the conversion factors of 7.7 pg DNA per chondrocyte, [21] and lo-'" g dry weight per chondrocyte [8]. The amount of GAG was determined spectrophotometrically after reaction with dimethylmethylene blue dye using bovine chondroitin sulfate in
B. Obradoilic et al. I Journal
of
Orthopaedic Research I 9 (2001) 1089-1097
1091
(a) Engineered constructs Chondrocvtes
u Scaffold
V
Construct cultivation (5 weeks)
Seeding (5 days)
Engineered constructs
(b) Construcffexplant composites
n
Central discs
Outer rings
Composites
Composite integration (1 - 8 weeks)
Fig. 1. Model system. Engineered constructs (cultured for 5 f 1 days or 5 f 1 weeks) or explants (untreated or trypsin treated cartilage) in form of 5 mm in diamater x 2 mm thick discs were sutured into ring-shaped explants (untreated or trypsin treated cartilage) to form composites that were cultured for an additional 1-8 weeks in bioreactors and evaluated biochemically, histologically and mechanically. phosphate buffer as a standard [6].The total amount of collagen was determined from the hydroxyproline content after acid-hydrolysis (6 N HC1 at 115°C for 18 h) and reaction with p - dimethylaminobenzaldehyde and chloramine-T [35] using a hydroxyproline to collagen ratio of 0.1 [17]. Histological analyses
Histological analyses were done for 5 day constructs, 5 week constructs, treated and untreated explant discs and rings, and disc-ring composites (at the time of preparation, and after 1 , 2 , 4 and 8 weeks of cultivation). Samples were fixed in 10% neutral buffered formalin, embedded in paraffin and sectioned (5 pm thick) through the center, either in the axial direction (cross-sections) or in the transverse plane (en face). Sections were stained with hematoxylin and eosin (H&E) for cells or with safranin-O/fast green for GAG. For immunohistochemical assessment, sections were incubated for 30 min at 37°C with 1 mg/ ml testicular hyaluronidase (Sigma-Aldrich, St. Louis MO), for 15 min at 25°C with normal goat serum (Sigma-Aldrich, St. Louis MO) diluted 1:lO in PBS, and for 1 h at 25°C with a primary monoclonal antibody against bovine collagen type I (Biodesign International, Kennebunk, ME), and stained using an alkaline phosphatase kit (Dako LSAB, Carpenteria, CA). Cell density in the integration region of composites was measured using H&E stained transverse sections ( n = 1-2 per data point) of tissue composites. Black and white images ( n = 15-20 per sample, each with an area of 0.08 mm2) were acquired by a solid-state CCD camera (Hitachi) mounted on an inverted microscope (Nikon ,Diaphot), digitized by a CG-7 frame grabber (Scion Corp., Frederick, MD) and analyzed using the Scion-Image program (Scion Corp., Frederick, MD). In each image, the number of cells was determined by counting automatically all objects that were darker than the background after the polymer fibers were excluded. The average number of counted cells per unit of image area was used as an indicator of cell density in the integration area.
0.15 M phosphate buffered saline (PBS, pH 7.4) supplemented with 100 U/cm3 penicillin and 10 pg/cm3 streptomycin, and each disk was mounted in an electrically insulated cylindrical confining chamber [7]. The chamber was mounted in a servo-controlled Dynastat mechanical spectrometer (IMASS, Hingham, MA) and the specimens were compressed at sequential increments of 10% strain up to a maximum of 40% strain, in the range where equilibrium stress varied linearly with applied strain. After stress relaxation, the equilibrium stress was measured and plotted against applied strain; the equilibrium modulus was determined from the slope of the best linear regression fit. Adhesive strength at the disk-ring interface of tissue composites ( n = 4 per data point) was assessed as the stress required to fracture the integration site by a plunger applied to the disk surface during a pushthrough test. The sample was placed in a custom-designed chamber between an annular main body covered with a fine sandpaper and an annular support ring, and fastened by a top housing such that the outer explant ring of composite was securely clamped providing mechanical no-slip. The plunger with exactly the same diameter as the central disk was connected to a load cell and the main body was advanced toward it at a rate of 0.5 m d m i n to engage the interface against the plunger. The load was measured until the plunger was forced through the disk-ring interface; no apparent deformation of the component discs and rings was observed at the time of failure. The adhesive strength of the composite was evaluated as the force at ultimate failure per unit of the interfacial area. Statistical analysis
Statistical analysis was performed by one-way analysis of variance (ANOVA) in conjunction with Tukey’s post hoc test for multiple comparisons using SPSS 8.0 for PC (SPSS, Chicago).
Mechanical testing
Results
Equilibrium moduli of central disks were determined as previously described [32]. In brief, 3 mm diameter by 2 mm thick disks were harvested from central regions of constructs or explants (n = 3 4 samples per group), equilibrated for 10-15 min at room temperature in
Fig. 2 shows representative sections of the component tissues and the resulting composites. 5 day constructs appeared largely inhomogeneous in many respects
1092
B. Ohradocic et al. I Journal of Orthopaedic Research 19 (2001) 1089-1097
Experimental groups:
Central disc
Outer ring
1 2 3
5 day construct 5 week construct Intact explant
Intact explant Intact explant Intact explant
4
5 day construct 5 week construct Treated explant
Treated explant Treated explant Treated explant
Fig. 2. Experimental groups. Six groups of composites were established using discs made of ( a ) 5 day constructs, (b) 5 week constructs, (c) untreated or (d) trypsin-treated explants, in conjunction with untreated or trypsin-treated explant rings; stain: safranin-0. Representative appearance of a composite shown for group 1 en face (e) and in cross-section (f) after 2 weeks of bioreactor cultivation; stain: safranin-0; scale bar: 1 mm. The table summarizes component tissues in the six groups of composites
(Fig. 2(a)). The construct periphery contained predominantly mature single chondrocytes embedded in extracellular matrix with GAG concentration that was markedly higher than in the inner tissue phase but lower than in cartilage explants; a monolayer of densely packed rounded cells was present at the surface. The inner tissue phase contained areas with immature cells starting to produce cartilaginous matrix, thereby separating themselves from the polymer fibers and each other. In contrast, 5 week constructs (Fig. 2(b)) appeared cartilaginous over their entire cross-sections. In the inner tissue phase, polymer fibers were partially degraded and immature cells were replaced by mature chondrocytes. A 30-100 pm thick layer at the construct periphery contained immature elongated cells and little GAG, and resembled the inner layer of native perichondrium. Untreated explants (Fig. 2(c)) consisted of mature chondrocytes, either single or in isogenous groups, surrounded by cartilaginous matrix with the highest concentration of GAG of the four groups. No characteristic cells are present at the explant surfaces because they were cored from the middle sections of full thickness cartilage. Empty channels correspond to blood vessels in immature cartilage. Trypsin treated explants (Fig. 2(d))consisted of an external zone containing little or no GAG, and an internal zone with GAG concentration comparable to that in untreated explants. The initial compositions are shown in Table 1 for 5 day constructs (groups 1 and 4), 5 week constructs (groups 2 and 5), intact explant rings (groups 1-3), trypsin treated explant discs and rings with 0.6 mm thick peripheral zone (group 6), and trypsin treated explant rings with 1.2 mm thick peripheral zone (groups 4-5).
Over 4 weeks of bioreactor cultivation, the wet weights of composite discs and rings increased in all groups by approximately two times (from 50 f 10 to 100 f 24 mg per disc and from 1 4 9 f 9 to 2 7 6 f 9 9 mg per ring, n = 18). GAG fraction increased in constructs and treated explants and decreased in untreated explants, and the collagen fraction increased in constructs and decreased in explants. Table 1 summarizes the biochemical compositions of composite discs and rings in all six experimental groups after 4 weeks in culture; the same trends were maintained after 8 weeks in culture (data not shown). The 5-day construct discs (groups 1 and 4) had a high cellularity, contained small amounts of GAG and collagen (Table 1) and were too fragile to allow the measurement of mechanical properties. The 5 week construct discs (groups 2 and 5) had normal cellularity, relatively higher but subnormal wet weight fractions of GAG and collagen (Table l), and an equilibrium modulus of 2 2 4 f 4 4 kPa. Untreated and treated explant discs had normal cellularity and collagen content, markedly different wet weight fractions of GAG (5.7 k 0.06 and 3.66 f 0.18, respectively (Table 1)) and equilibrium moduli of 928 f 30 kPa. After 4 weeks of composite cultivation, disc cellularities reached a plateau at a normal level, while GAG and collagen fractions remained subnormal in both 5 day and 5 week construct discs, and were at normal level in both untreated and treated explant discs. The outer rings had normal cellularity in all groups and at all time points. GAG fractions in explant rings became comparable for all groups after 4 weeks of composite cultivation (Table 1). The corresponding collagen fractions decreased in
B. Obradovic et al. I Journal of Orthopaedic Research 19 (2001) 1089-1097
1093
Table 1 Biochemical compositions of composite discs and ringsa Experimental group (see Fig. 2)
Cells (% wet weight)
GAG (% wet weight)
Collagen (YOwet weight)
Central disc
Outer ring
Central disc
Outer ring
Central disc
Outer ring
Initial composites (at the time of preparation) I 1.51 ! ~ 0 . 1 4 ~ 1 ~ 0.69f0.06 0.80 f0.13 0.69 f 0.06 2 3 0.85 f0.09 0.69 f 0.06 4 1.51 f 0.14b,d 0.68 f 0.10 5 0.80 f 0.13 0.68 f 0.10 0.75 f 0.03 6 0.98 f0.1 1
1.88 fO . l l d 2.54 f 0.09d 5.70 f0.06b 1.88f0.11 2.54 f 0.09 3.66 f0.18
6.42 i0.74 6.42 f 0.74 6.42 f0.74 1.34 i0.29h 1.34 f0.29b 3.95 f 0.60
1.10 fO.OSd 2.34 f0.35* 9.54 f 2.2b 1.10f 0.06d 2.34 f0.35d 8.93 f 0.31b
11.1 f 1.6 ll.lf1.6 11.1 f 1 . 6 11.8f1.5 11.8f 1.5 10.2f 1.3
Cultured composites (4 weeks in rotating bioreactors) 1 1.00 f 0.08'1~ 0.63 f 0.10 2 0.76 & 0.06 0.64 f0.08 3 0.65 f0.06 0.58 f 0.04 4 1.10 f 0.08' 1.00 f 0.05 5 0.96 f 0.01 1.09 f0.08 6 0.84 f0.07 0.76 f 0.06b
2.81 f 0.38 3.27 f0.18 5.17 f 0.43b 2.99 f 0.31 2.12 f 0.26 5.23 f 1.02b)d
3.83 f 0.34' 4.96 i0.70 5.82 f0.77 3.76 f.O.3lc 3.15 f 0.10" 5.65 f 0.50Czb
2.60 f 0.23d 3.06 f 0.12 5.97 f 1.lh 2.97 f 0.1Sd 3.09 f 0.17d 7.06 f 2.37b
7.61 f 0 . 2 1 5.86f0.89' 5.83 f0.77' 9 . 2 f 1.4 11.1~k3.6~ 7.18f2.1
"Statistically significant differences (P< 0.05, n = 3). ~ other ~ . groups; same time point. "4 weeks vs. initial; same group. Disc vs. ring; same group; same time point. b
rings from all composite groups except for groups 4 and 5 where it was maintained at the initial level. Integration of 5 day constructs and untreated explant rings involved the progressive formation of cartilaginous tissue at the disc-ring interface (Fig. 3). After 1 week of composite cultivation (Figs. 3(a) and (d)), the central
disc and the region at the disc-ring interface consisted of immature tissue with a high density of elongated cells, low concentration of GAG and many undegraded polymer fibers. Only a few small islands of tissue containing rounded cells surrounded by GAG indicated the maturation of the forming tissue. Empty spaces were
Fig. 3. Integration patterns. (a)-(f) Effects of composite cultivation time. Disc-ring interfaces of group 1 composites, shown in cross-section at low magnification (a)-(c) and high magnification (d)qf). Composite cultivation time: (a) and (d) 1 week, (b) and (e) 2 weeks, (c) and (f) 4 weeks. Stain: safranin 0;scale bar: (a)-(c) 1 mm or ( d H f ) 100 pn; ring on left and disc on right of each image. (g) and (h) Expression of GAG and type I collagen. Low magnification sections of disc-ring interface stained with (8) safranin-0 and (h) immunohistochemically for collagen type I; scale bar: 1 mm. PC: proliferating cells, IC: immature cartilaginous tissue, MC: mature cartilaginous tissue.
1094
B. Ohradovic et al. I Journal of Orthopaedic Research 19 (2001) 1089-1097
still clearly visible at the disc-ring interface as well as in the inner regions of the central disc. After 2 weeks of composite cultivation (Figs. 3(b) and (e)), the inner disc contained large interconnected regions of immature and maturing tissues described above, and mature cartilage characterized by round cells within clearly defined lacunae and an organized matrix rich in GAG. The presence of immature tissue within 100-200 pni of the disc-ring interface, at the outer surfaces and around empty spaces (Fig. 3(e))was associated with the actively dividing cells able to fill the gaps and create a provisional tissue matrix containing high density of proliferating cells and little GAG. After 4 weeks in culture (Figs. 3(c) and (Q),only small areas of immature tissue were present at the interface, and even those areas contained rounded cells in their lacunae and a cartilaginous matrix. The central disc consisted of mature articular cartilage containing round cells, ECM rich in GAG and only occasional polymer fibers. Between weeks 4 and 8 in culture, the composites remained largely unchanged from the light microscopic standpoint. The progressive formation of cartilaginous tissue over the course of composite cultivation was associated with the complementary expression of GAG and type I collagen as follows. The region at the disc-ring interface containing proliferating cells and the regions of immature tissue within the central disc stained strongly with type I collagen antibody, but not with safranin-0 (regions PC and IC, respectively, in Figs. 3(g) and (h)) and type I1 collagen (data not shown). In contrast, the regions of more mature cartilaginous tissue within the outer ring and central disc stained strongly with safranin-0 and type I1 collagen antibody, but did not express type I collagen, except around blood vessels in cartilage explants (region MC in Figs. 3(g) and (h)). Maturing cartilage had an intermediate behavior, staining both for GAG and type I1 collagen (but not as strongly as cartilage) and type I collagen (but not as strongly as immature tissue).
Fig. 4 shows representative histological sections of composites after 4 weeks of cultivation. As compared to the other groups, the disclring interface of group 1 composites contained a 150-200 pm thick zone of proliferative tissue with a relatively high cell density (1070 f 300 cells/mm2) and only a small amount of GAG (Fig. 4(a)). The cells appeared to be primarily rounded chondrocytes in their characteristic lacunae, with a small fraction of elongated cells. No such reparative zone was observed in composites made using 5 week constructs or cartilage explants (Figs. 4(b) and (c)), where the tissue within the central disc was cartilaginous even at the earliest time points. Gaps between the central disc and outer ring remained at the 4 week time point, and did not contain proliferating cells, but rather an acellular tissue matrix (640 f 130 and 430 90 cells/ mm2 for groups 2 and 3 composites, respectively). Trypsin treatment of cartilage explants induced chondrocytes to proliferate and form isogenous groups of 2-3 cells which rapidly produced GAG to replenish the matrix lost during the trypsin treatment. The integration patterns were markedly affected by trypsin treatment only in group 4 composites based on 5 day constructs but not in group 5 and group 6 composites based on 5 week constructs and treated explants, respectively (Fig. 4(d vs a)). In group 4 composites, the integrating area stained more intensely with safranin-0 than in the corresponding group 1 composites. In particular, as the tissue at the interface matured, cartilaginous tissue buds began to extend from the construct disc into the treated explant ring creating the appearance of an undulated interface, a phenomenon not observed in composites made using untreated explants. The measured adhesive strength of composites (Fig. 5) was consistent with the integration patterns observed for different experimental groups. The adhesive strengths of composites based on 5 week constructs, untreated or treated cartilage explants (groups 2, 3, 5, and 6 ) were in the range of 80-160 kPa. In contrast, the
Fig. 4. Integration patterns: effects of tissue composition. Representative cross-sections of integration interfaces in six groups of composites: (a) group 1, (b) group 2, (c) group 3, (d) group 4, (e) group 5 and (0 group 6 . Cultivation time: 4 weeks: stain: safranin-0; scale bar: 200 pm; disc on the top, ring on the bottom.
B. Obradouic et al. I Journal of Orthopuedic Research 19 (2001 I 1089-1097
500
*
-1
T
i6 1
2
3
4
5
6
Group Fig. 5. Adhesive strength of the integration interface. Adhesive strength of the integration interface is shown as a function of experimental group. Data represent the average iS.D. of n = 3 4 independent samples. (*) significantly different from all other experimental groups.
adhesive strengths of composites prepared using central disks made of 5 day constructs and either untreated or treated outer rings were, respectively, 254 f 66 kPa (group 1) and 384 f 63 kPa (group 4). Adhesive strength was therefore significantly higher for group 4 composites as compared to all other groups.
Discussion We report controlled studies of the formation of the tissue bond between engineered and native cartilage in tissue composites cultured for up to 8 weeks in bioreactors, and demonstrate that the integrative properties depend on the developmental stage of engineered constructs and the proteoglycan removal from the adjacent cartilage. The highest adhesive strength of the integration interface was observed for composites made from immature constructs and trypsin treated explants, and could be attributed to the formation of cartilaginous tissue by densely populated cells. Bioreactor studies of tissue composites can thus help relate the integrative properties of engineered constructs to the stage of their development. The integrative properties, evaluated by patterns of tissue remodeling and adhesive strength of the integrating interface, was generally higher for immature than for more mature tissues. Immature constructs, cultured for 5 days, had poor initial mechanical properties but integrated more rapidly than either maturing constructs cultured for 5 weeks, or cartilage explants. The integration of immature constructs with the adjacent cartilage was associated with the presence of proliferating cells and the formation of cartilaginous tissue bond (Figs. 3 and 4(a)) in contrast to the secretion of matrix components into a relatively acellular interface
1095
region of composites containing more mature constructs or cartilage explants (Figs. 4(b) and (c)). The observed integration patterns correlated with the adhesive strength of the tissue interface, which was markedly higher for immature constructs than for either mature constructs or cartilage explants (Fig. 5). This suggests that the integration patterns involving only the deposition of ECM proteins (i.e., in composites based on 5 week constructs or cartilage explants) result in lower adhesive strengths than the integration patterns which involve active tissue remodeling by proliferative cells (i.e. in composites based on 5 day constructs). Previous studies of the integrative properties of cartilage explants cultured in vitro for 3 weeks and assessed mechanically in a tensile single lap configuration [25] also reported that the adhesive strength depended on the presence on viable cells at tissue surfaces. A recent study of in vitro engineered cartilage/bone composites [28] also demonstrated that the integration at the cartilage/bone interface was better in composites consisting of immature (1 week old) than mature (4 week old) constructs. The developmental stage of the tissue determined the compositions of both the central discs and outer rings in tissue composites (Table 1, Fig. 4). Over 8 weeks of cultivation, the compositions of discs and rings approached each other in all groups, which is likely to play a role in functional response of the tissue graft to physiological loads. The associated changes in the concentrations of tissue components were higher for composites made using immature than mature tissues, a trend consistent with previously observed decreases in construct synthesis rates of GAG and collagen with cultivation time [ 1 11. Likewise, low concentrations of GAG in central discs of group 5 composites might be attributed to relatively low synthesis rates and the loss of newly synthesized GAG into the treated explant rings. These observations indicate that the compositions of the adjacent tissues depend on one another, and on developmental stage of the central discs as well as trypsin treatment of the outer rings. Partial removal of the tissue GAG can enhance cartilage remodeling. Proteoglycans are known to inhibit cell adhesion [26], and this effect can be reversed by treating cartilage explants with proteolytic enzymes [36]. Partial removal of proteoglycans by enzyme treatment of the chondral surface and local introduction of mitogenic factors were reported to improve cell recruitment from the synovial membrane, defect coverage and repair [19,20]. It has been long known that the removal of ECM induces chondrocytes to resume DNA synthesis and proliferate [1,2,18]. Under the conditions of the present study, trypsin treatment resulted in partial removal of GAG without significant change in collagen concentration. The treatment affected the integration patterns in composites made using immature (5 day) constructs (Figs. 4(a) and (b)), but had no apparent
1096
cdic Reseurch I 9 (2001) 1089-1097 B. Obradouic et af. I Journul of Orthopa$
effect on composites based on 5 week constructs or cartilage explants (Fig. 4(c)-(f)). The presence of proliferating cells was key for the progressive remodeling of the integrating interface which involved “budding” of the newly synthesized tissue into the trypsin treated explants (Fig. 4(b)). Devitalized lamb cartilage explants seeded with viable chondrocytes and implanted in nude mice produced large buds of new cartilage penetrating into the devitalized tissue [24]. Relatively small buds observed in our study were probably due to better preserved structure of trypsin treated explants as compared to devitalized tissue. The highest adhesive strength of the tissue interface of composites based on 5 day constructs and trypsin treated explants suggests that GAG removal from the adjacent tissue may further increase the integrative properties of engineered cartilage grafts. Possible mechanisms of action may involve the enhancement of cell migration and proliferation by increasing the permeability of the adjacent tissue, which is consistent with little if any effect of trypsin treatment on integration patterns of 5 week constructs and cartilage explants that did not involve the presence of proliferating cells. The progressive formation of cartilaginous tissue at the disc-ring interface involved the synthesis and ‘assembly of extracellular matrix by densely populated cells and the shift in gene expression away from type I collagen. Initially, discs made of 5 day constructs contained high density of rapidly proliferating elongated cells that resembled immature mesenchymal cells within a loose matrix that contained very little GAG and stained positively for type I collagen (Figs. 3(g)-(j)). Processes at the disc-ring interface resembled some of the embryonic events at the growth plate, where “early” cartilage produced by limb mesenchyme is also composed of predominantly type I collagen [16]. The appearance of type I1 collagen in stage 23-24 limb buds [30] coincided with the differentiation of the condensed mesenchymal cells into chondroblasts [ 161. Likewise, the progressive formation of cartilaginous matrix in the central disc and at the disc-ring interface coincided with the lack of expression of type I collagen. As a result, the majority of the tissue in the central discs of composites based on 5 day constructs and explant rings matured into well organized cartilaginous tissue matrix by 4-8 weeks in culture. In summary, bioreactors can provide controlled environment for studies of factors affecting the integration of engineered and native cartilage with the adjacent tissue without systemic effects and variability that are inherent for in vivo studies. Bioreactor studies can thus help in the planning and interpretation of complementary in vivo studies addressing the entirety of biochemical and physical regulatory signals that modulate cartilage function in an articular joint. The above findings suggest that the most important factor for inte-
grative tissue repair is the presence of biosynthetically active cells, capable of proliferating, filling the gaps at tissue interface, and progressively forming cartilaginous tissue. The duration of bioreactor cultivation for engineered constructs could be selected to achieve a desired combination of compressive stiffness (which increases with time in culture) and integrative properties (which decrease with time in culture). The use of enzymes or additional chondrogenic cells at the time of implantation may further enhance the integrative tissue repair. For any given application, specific requirements are likely to further depend on cell source, construct geometry and loading conditions, and need to be determined in coordinated in vitro and in vivo studies.
Acknowledgements The authors would like to thank Li Zeng for technical help. The research was funded by the National Aeronautics and Space Administration (NASA, Grant NAG9-836).
References [l] Abbott J, Holtzer H. The loss of phenotypic traits by differentiated cells. J Cell Biol 1966;28:473-87. [2] Abbott J, Holtzer H. The effect of 5-bromodeoxyuridine on cloned chondrocytes. PNAS 1968;59:1144-51. [3] Buckwalter JA, Mankin HJ. Articular cartilage repair and transplantation. Arthritis Rheum 1998;41:133142. [4] Buschmann MD, Gluzband YA, Grodzinsky AJ, Kimura JH, Hunziker EB. Chondrocytes in agarose culture synthesize a mechanically functional extracellular matrix. J Orthop Res 1992;10:745-58. [5] Chen FS, Frenkel SR, DiCesare PE. Repair of articular cartilage defects: Part 11. Treatment options. Am J Orthop 1999;28:88-96. [6] Farndale RW, Buttle DJ, Barrett AJ. Improved quantitation and discrimination of sulphated glycosoaminoglycans by the use of dimethylmethylene blue. Biochim Biophys Acta 1986;883:173-7. [7] Frank EH, Grodzinsky AJ. Cartilage electromechanics. I. Electrokinetic transduction and the effects of electrolyte pH and ionic strength. J Biomech 1987;20:615-27. [8] Freed LE, Vunjak-Novakovic G, Biron RJ, Eagles DB, Lesnoy DC, Barlow SK, Langer R. Biodegradable polymer scaffolds for tissue engineering. Bio/Technology 1994;12:689-93. [9] Freed LE, Vunjak-Novakovic G. Cultivation of cell-polymer tissue constructs in simulated microgravity. Biotech Bioeng 1995;46:306-13. [lo] Freed LE, Langer R, Martin I, Pelli‘s N, Vunjak-Novakovic G. Tissue engineering of cartilage in space. Proc’Natl Acad Sci USA 1997;94:13885-90. [l I] 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. [12] Freed LE, Vunjak-Novakovic G. Culture of organized cell communities. Adv Drug Del Rev 1998;33:15-30. [I31 Freed LE, Martin I, Vunjak-Novakovic G. Frontiers in tissue engineering: in vitro modulation of chondrogenesis. Clin Orth Re1 Res 1999;367S:S4&58.
B. Obradovic et al. I Journal of Orthopaedic Research 19 (2001) 1089-1097
[I41 Freed LE, Vunjak-Novakovic G. Tissue engineering of cartilage. 2nd ed. In: Bronzino J, editor. Biomedical engineering handbook. Boca Raton: CRC Press;2000, p. 1-26 [Chapter 1241. [I 51 Freed LE, Vunjak-Novakovic G. Tissue culture bioreactors: chondrogenesis as a model system. 2nd ed. In: Lanza RP, Langer R, Austin JV, editors. Principles of tissue engineering. New York: Academic Press; 2000, p. 143-56 [Chapter 131. [I61 Hall H.K. Developmental and cellular skeletal biology. New York:Academic Press; 1978, p. 134-5, 164-70. [17] Hollander AP, Heathfield TF, Webber C, Twata Y , Bourne R, Rorabeck C, Poole AR. Increased damage to type I1 collagen in osteoarthritic articular cartilage detected by a new immunoassay. J Clin Invest 1994;93:1722-32. [18] Holtzer H, Abbott J, Lash J, Holtzer S. Dedifferentiation of cartilage cells. PNAS 1960;46:153342. [19] Hunziker EB, Rosenberg LC. Repair of partial-thickness defects in articular cartilage: cell recruitment from the synovium. J Bone Joint Surg A 1996;78:721-33. [20] Hunziker EB, Kapfinger E. Removal of proteoglycans from the surface of defects in articular cartilage transiently enhances coverage by repair cells. J Bone Joint Surg B 1998;80:14450. [21] Kim YJ, Sah RL, Doong JYH, Grodzinsky AJ. Fluorometric assay of DNA in cartilage explants using Hoechst 33258. Anal Biochem 1988;174:168-76. [22] O’Driscoll S. The healing and regeneration of articular cartilage. J Bone Joint Surg A 1998;80:1795-812. [23] Paget J. Healing of cartilage. Clin Orthop 1969;64:7-8. Reprinted from Lecture XII: Healing of injuries in various tissues. In: Lectures on surgical pathology. London, p. 1-262, 1853. [24] Peretti GM, Randolph MA, Caruso EM, Rossetti F, Zaleske DJ. Bonding of cartilage matrices with cultured chondrocytes: an experimental model. J Orthop Res 1998;16:89-95. [25] Reindel ES. Ayroso AM, Chen AC, Chun DM, Schinagl RM, Sah RL. Integrative repair of articular cartilage in vitro: adhesive strength of the interface region. J Orthop Res 1995;13:751-60. [26] Rich AM, Pearlstein E, Weissmann G, Hoffstein ST. Cartilage proteoglycans inhibit fibronectin-mediated adhesion. Nature 1981:293:2%6.
1097
[27] Sah RLY, Kim IJ, Doong JYH, Grodzinsky AJ, Plaas AHK, Sandy JD. Biosynthetic response of cartilage explants to dynamic compression. J Orthop Res 1989;7:619-36. [28] Schaefer D, Martin I, Shastri P, Padera RF, Langer R, Freed LE, Vunjak-Novakovic G. In vitro generation of osteochondral composites. Biomaterials 2000;21(24):2599-606. [29] Shortkroff S, Barone L, Hsu H-P, Wrenn C, Gagne T, Chi T, et al. Healing of chondraland osteochondral defects in a canine model: the role of cultured chondrocytes in regeneration of articular cartilage. Biomaterials 1996;17:147-54. [30] von der Mark H, von der Mark K, Gay S. Study of differential collagen synthesis during the development of the chick embryo by immunofluorescence. I. Preparation of collagen type I and type I1 specific antibodies and their application to early stages of the chick embryo. Dev Biol 1976;48:23749. [31] Vunjak-Novakovic G, Obradovic B, Martin I, Bursac PM, Langer R, Freed LE. Dynamic cell seeding of polymer scaffolds for cartilage tissue engineering. Biotechnol Prog 1998;14:193-202. [32] Vunjak-Novakovic G, Martin I, Obradovic B, Treppo S, Grodzinsky AJ, Freed LE. Bioreactor cultivation conditions modulate the composition and mechanical properties of tissue-engineered cartilage. J Orthop Res 1999;17:130-8. [33] Wakitani S, Kimura T, Hirooka A, Ochi T. Yoneda M, Yasui N, Owaki H, Ono K. Repair of rabbit articular surfaces with allograft chondrocytes embedded in collagen gel. J Bone Joint Surg B 1989;71:7&80. [34] Wakitani S, Goto T, Pineda SJ, Young RG, Mansour JM, Caplan AI, Goldberg VM. Mesenchymal cell-based repair of large, fullthickness defects of articular cartilage. J Bone Joint Surg A 199%76:579-92. [35] Woessner JF. The determination of hydroxyproline in tissue and protein samples containing small proportions of this imino acid. Archiv Biochem Biophys 1961;93:440-7. [36] Yamagata M, Suzuki S, Akiyama SK, Yamada KM, Kimata K. Regulation of cell-substrate adhesion by proteoglycans immobilized on extracellular substrates. J Biol Chem 1989;264:8012-8.
ELSEVIER
Journal of Orthopaedic Research 19 (2001) 1089-1097 www.elsevier.com/locate/orthres
Integration of engineered cartilage S. Treppo B. Obradovic b, I. Martin a, R.F. Padera G. Vunjak-Novakovic a,* a
a,
L.E. Freed
a,
Diuision of Health Sciences and Techtiology, Massachusetts Institute of Technology, E2.5 Room 330, 45 Carleton Street, Cirmbridge, M A 02139, USA Depurtment of Chemiral Engineering, Tufts Uniiiersity, Medford, M A 02155, USA Harvard Medical School. Boston, M A 02115, U S A
Received 3 April 2000; accepted 31 January 2001
Abstract The structure and function of cartilaginous constructs, engineered in vitro using bovine articular chondrocytes, biodegradable scaffolds and bioreactors, can be modulated by the conditions and duration of tissue cultivation. We hypothesized that the integrative properties of engineered cartilage depend on developmental stage of the construct and the extracellular matrix content of adjacent cartilage, and that some aspects of integration can be studied under controlled in vitro conditions. Disc-shaped constructs (cultured for 5 i 1 days or 5 1 weeks) or explants (untreated or trypsin treated cartilage) were sutured into ring-shaped explants (untreated or trypsin treated cartilage) to form composites that were cultured for an additional 1-8 weeks in bioreactors and evaluated biochemically, histologically and mechanically (compressive stiffness of the central disk, adhesive strength of the integration interface). Immature constructs had poorer mechanical properties but integrated better than either more mature constructs or cartilage explants. Integration of immature constructs involved cell proliferation and the progressive formation of cartilaginous tissue, in contrast to the integration of more mature constructs or native cartilage which involved only the secretion of extracellular matrix components. Integration patterns correlated with the adhesive strength of the disc-ring interface, which was markedly higher for immature constructs than for either more mature constructs or cartilage explants. Trypsin treatment of the adjacent cartilage further enhanced the integration of immature constructs. 0 2001 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved.
*
Introduction It has long been known that mature articular cartilage has a limited capacity for regeneration after degeneration or injury [23]. Methods used clinically to enhance natural repair of articular surfaces include modifications of the damaged surface (chondral shaving with debridement, abrasion arthroplasty, subchondral drilling or microfracturing of the subchondral plate) and transplantation of periosteal, perichondral or osteochondral autografts [5]. In most cases, these methods temporarily relieve symptoms and induce an initial hyaline-like repair, but the repair tissue eventually degenerates to fibrocartilage and the symptoms return. None of the currently available methods can predictably restore a durable articular surface [3].
*Corresponding author. Tel.: +I-617-452-2593; fax: +I-617-2588827. E-mail address: [email protected] (G. Vunjak-Novakovic).
Novel approaches to cartilage repair include the use of autologous or allogenic chondrocytes or chondroprogenitor cells injected at the defect site, alone or in conjunction with collagen gels, fibrin, ceramic, and degradable or non-degradable synthetic polymers [22]. Although these approaches are promising, long-term maintenance of regenerated hyaline tissue under conditions of normal joint loading was variable and inconsistent [5]. Systematic studies are needed to determine the specific factors (e.g., cells, biomaterials, enzymes, mechanical loading) governing the integration of transplanted cells or tissues into the site of a chondral or osteochondral defect. Although cell-based grafts have been studied in animal models with respect to the choice of carrier and the methods of graft fixation [22,24,29,33,34], the criteria for graft selection and the mechanisms of graft-host integration are not yet well understood. The variability of the in vivo environment further complicates evaluation of the results. Cartilage tissue engineering has been motivated by the need to replace lost or damaged tissue with an
0736-0266/01/$ - see front matter 0 2001 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved. PII: S 0 7 3 6 - 0 2 6 6 ( 0 1 ) 00 0 3 0 - 4
1090
B. Obradovic et al. I Journal of Orthopaedic Research 19 (2001) 1089-1097
already structurally and mechanically functional implant that can be created in vitro using chondrocytes or chondroprogenitor cells in conjunction with biomaterials [4,12-151. We have previously demonstrated that cartilaginous constructs engineered using articular chondrocytes, biodegradable polymer scaffolds and bioreactors had structure (biochemical composition, histomorphology) and function (mechanical behavior, biosynthesis rates) that depended on the conditions and duration of bioreactor cultivation [32]. After 6 weeks in culture, constructs were cartilaginous throughout their volume [l 11, and had wet weight fractions of glycosaminoglycan (GAG) and collagen and equilibrium moduli of 78%, 43% and 25% of the respective values measured for freshly explanted cartilage 1321. After prolonged (7 months) cultivation, wet weight fractions of GAG and equilibrium moduli of engineered constructs were comparable or exceeded values measured for cartilage [lo]. In the present work, the integration of engineered constructs with native cartilage was studied in disdring composites cultured in bioreactors for up to 8 weeks. We hypothesized that the integrative properties of engineered constructs can be modulated by the duration of bioreactor cultivation (which determines cell biosynthetic activity and construct structure, composition, and mechanical behavior) and by trypsin treatment of the adjacent cartilage (which determines tissue concentration of GAG).
Materials and methods Cartilaginous constructs
Cartilage was engineered as previously described [10,32] using articular chondrocytes, biodegradable polyglycolic acid (PGA) scaffolds and bioreactors. Full thickness articular cartilage was harvested from the femoropatellar grooves (FPG) of 2-3 week old bovine calves within 8 h of slaughter. Chondrocytes were isolated using type I1 collagenase (Worthington, Freehold, NJ) and resuspended in culture medium (Dulbecco's Modified Eagle Medium (DMEM) containing 4.5 gA glucose and supplemented with 10% fetal bovine serum, 10 mM N-2hydroxyethylpiperazine "-2-ethane sulfonic acid (HEPES), 50 Ulml penicillin, 50 &ml streptomycin, 0.1 mM non-essential amino acids, 0.4 mM proline, 50 pg/ml ascorbic acid, 0.5 pg/ml fungizone) [4]. Scaffolds were fibrous disks, 5 mm in diameter x 2 mm thick, made of biodegradable PGA by Albany International (Mansfield, MA) as a highly porous mesh with a bulk density of 65 mg/cm3 and a void volume of 97% [8]. Scaffolds were dynamically seeded with freshly isolated chondrocytes as previously described [31], using 12 scaffolds per flask in 120 cm3 of cell suspension containing 5 x los cellslcm3 and stirred at 50 rpm. Gas exchange was provided by surface aeration (10% C 0 2 in humidified air). After 5 i 1 days of seeding, constructs were either harvested and used for composite preparation ( 5 day constructs) or cultured in bioreactors for an additional 5 f 1 weeks (5 week constructs). Twelve constructs per bioreactor were cultured in 110 cm3 volume annular space, as previously described [9,15]. Each bioreactor was rotated as a solid body around the horizontal axis; the rotation rate was adjusted to keep constructs freely settling in rotating flow, and gradually increased over time to accommodate the increase in construct size and weight. Gas exchange was provided by diffusion
through the inner cylinder. Culture medium was completely replaced after seeding, and then by 50% vlv every other day for the duration of cultivation. Cartilage explants
Natural cartilage plugs (10 mm in diameter) were harvested from cartilage cores obtained from the medial and lateral ridges adjacent to the FPG using a dermal punch (Miltex Instruments, Lake Success, N Y ) . The thickness of these cores always exceeded 4.5 mm. Discs (10 mm diameter x 2 mm thick) were obtained from the middle portion of the cores after removing 1 nim thick slices from both the articular surface and the regon adjacent to the subchondral bone, and rinsed with phosphate buffered saline supplemented with 100 U/cm3 penicillin and 50 pg/cm3 streptomycin. Dermal punches were then used to core the 10 mm diameter discs into final explant rings (outer and inner diameters 10 and 5 mm, respectively, 2 mm thick, in all groups) and explant discs (5 mm diameter x 2 mm thick, in all groups). In some groups, explant rings and discs were treated by trypsin (cell culture quality, type I, from Gibco, 1% in PBS at 37"C), by evenly exposing all surfaces to the enzyme solution. The duration of incubation was determined empirically and set to either 10 or 20 min, to reproducibly remove GAG from a -0.6 or 1.2 mm thick peripheral zone, as assessed from safranin-0 stained histological sections. Incubation time was set to 10 min when both rings and discs were treated, and 20 min when only rings were treated, such that the total thickness of the treated zone at the tissue interface was -1.2 mm. Composites
Discs ( 5 mm in diameter x 2 mm thick in all groups) were press-fit into the rings (1015 mm in diameter x 2 mm thick in all groups) and sutured together using #5-0 silk surgical suture and cutting FS-2 needle, with two stitches positioned at a 90" angle from each other. The disc and ring were connected at points that were approximately 1 mm away from the integration interface (Fig. 1). Six groups of composites were established using four different component tissues: constructs cultured for 5 days or 5 weeks, untreated or treated explants, as follows. Group 1-3 composites had central discs that were 5 day constructs, 5 week constructs or untreated explants, respectively, and outer rings that were intact explant rings. Group 4 6 composites had central discs that were 5 day constructs, 5 week constructs or treated explants (0.6 mm thick peripheral zone), respectively, and outer rings that were treated explants (1.2 mm thick peripheral zone in groups 4 and 5; 0.6 mm thick peripheral zone in group 6 ) . Composites were cultured for 1-8 weeks in rotating bioreactors (n = 12 composites from one experimental group per bioreactor) as described above for engineered constructs. Final rotation rates were as high as 45 rpm. The experiments involved a total of 87 engineered constructs, 151 explants and 95 composites (12-24 per experimental group), using articular cartilage obtained from 21 bovine knee joints. For each experimental group, composites were sampled at timed intervals and analyzed biochemically (discs and rings from n = 3 composites per data point), histologically (n = 2-3 composites per data point), and mechanically (discs from n = 3 4 composites per data point to measure equilibrium modulus; n = 4 composites per data point to measure adhesive strength), as follows. Biochemical analyses
Composites were sampled at the time of preparation, after 4 and 8 weeks of cultivation, and separated into discs and rings along the integration interface using a surgical blade. The disc and ring could be distinguished in all cases by careful macroscopic inspection of the integration interface. Samples were frozen, lyophilized and digested with 0.125 pg/ml papain (Sigma, St. Louis, MO) in 100 mM phosphate buffer (PBS) with 10 mM EDTA and 10 mM cysteine for 15 h at 60°C [27], using 1 ml enzyme solution per 4-40 mg dry weight of the sample. The number of chondrocytes was obtained from the measured amount of DNA, using Hoechst 33258 dye and spectrofluorometer (PTI, South Brunswick, NJ) [21], based on the conversion factors of 7.7 pg DNA per chondrocyte, [21] and lo-'" g dry weight per chondrocyte [8]. The amount of GAG was determined spectrophotometrically after reaction with dimethylmethylene blue dye using bovine chondroitin sulfate in
B. Obradoilic et al. I Journal
of
Orthopaedic Research I 9 (2001) 1089-1097
1091
(a) Engineered constructs Chondrocvtes
u Scaffold
V
Construct cultivation (5 weeks)
Seeding (5 days)
Engineered constructs
(b) Construcffexplant composites
n
Central discs
Outer rings
Composites
Composite integration (1 - 8 weeks)
Fig. 1. Model system. Engineered constructs (cultured for 5 f 1 days or 5 f 1 weeks) or explants (untreated or trypsin treated cartilage) in form of 5 mm in diamater x 2 mm thick discs were sutured into ring-shaped explants (untreated or trypsin treated cartilage) to form composites that were cultured for an additional 1-8 weeks in bioreactors and evaluated biochemically, histologically and mechanically. phosphate buffer as a standard [6].The total amount of collagen was determined from the hydroxyproline content after acid-hydrolysis (6 N HC1 at 115°C for 18 h) and reaction with p - dimethylaminobenzaldehyde and chloramine-T [35] using a hydroxyproline to collagen ratio of 0.1 [17]. Histological analyses
Histological analyses were done for 5 day constructs, 5 week constructs, treated and untreated explant discs and rings, and disc-ring composites (at the time of preparation, and after 1 , 2 , 4 and 8 weeks of cultivation). Samples were fixed in 10% neutral buffered formalin, embedded in paraffin and sectioned (5 pm thick) through the center, either in the axial direction (cross-sections) or in the transverse plane (en face). Sections were stained with hematoxylin and eosin (H&E) for cells or with safranin-O/fast green for GAG. For immunohistochemical assessment, sections were incubated for 30 min at 37°C with 1 mg/ ml testicular hyaluronidase (Sigma-Aldrich, St. Louis MO), for 15 min at 25°C with normal goat serum (Sigma-Aldrich, St. Louis MO) diluted 1:lO in PBS, and for 1 h at 25°C with a primary monoclonal antibody against bovine collagen type I (Biodesign International, Kennebunk, ME), and stained using an alkaline phosphatase kit (Dako LSAB, Carpenteria, CA). Cell density in the integration region of composites was measured using H&E stained transverse sections ( n = 1-2 per data point) of tissue composites. Black and white images ( n = 15-20 per sample, each with an area of 0.08 mm2) were acquired by a solid-state CCD camera (Hitachi) mounted on an inverted microscope (Nikon ,Diaphot), digitized by a CG-7 frame grabber (Scion Corp., Frederick, MD) and analyzed using the Scion-Image program (Scion Corp., Frederick, MD). In each image, the number of cells was determined by counting automatically all objects that were darker than the background after the polymer fibers were excluded. The average number of counted cells per unit of image area was used as an indicator of cell density in the integration area.
0.15 M phosphate buffered saline (PBS, pH 7.4) supplemented with 100 U/cm3 penicillin and 10 pg/cm3 streptomycin, and each disk was mounted in an electrically insulated cylindrical confining chamber [7]. The chamber was mounted in a servo-controlled Dynastat mechanical spectrometer (IMASS, Hingham, MA) and the specimens were compressed at sequential increments of 10% strain up to a maximum of 40% strain, in the range where equilibrium stress varied linearly with applied strain. After stress relaxation, the equilibrium stress was measured and plotted against applied strain; the equilibrium modulus was determined from the slope of the best linear regression fit. Adhesive strength at the disk-ring interface of tissue composites ( n = 4 per data point) was assessed as the stress required to fracture the integration site by a plunger applied to the disk surface during a pushthrough test. The sample was placed in a custom-designed chamber between an annular main body covered with a fine sandpaper and an annular support ring, and fastened by a top housing such that the outer explant ring of composite was securely clamped providing mechanical no-slip. The plunger with exactly the same diameter as the central disk was connected to a load cell and the main body was advanced toward it at a rate of 0.5 m d m i n to engage the interface against the plunger. The load was measured until the plunger was forced through the disk-ring interface; no apparent deformation of the component discs and rings was observed at the time of failure. The adhesive strength of the composite was evaluated as the force at ultimate failure per unit of the interfacial area. Statistical analysis
Statistical analysis was performed by one-way analysis of variance (ANOVA) in conjunction with Tukey’s post hoc test for multiple comparisons using SPSS 8.0 for PC (SPSS, Chicago).
Mechanical testing
Results
Equilibrium moduli of central disks were determined as previously described [32]. In brief, 3 mm diameter by 2 mm thick disks were harvested from central regions of constructs or explants (n = 3 4 samples per group), equilibrated for 10-15 min at room temperature in
Fig. 2 shows representative sections of the component tissues and the resulting composites. 5 day constructs appeared largely inhomogeneous in many respects
1092
B. Ohradocic et al. I Journal of Orthopaedic Research 19 (2001) 1089-1097
Experimental groups:
Central disc
Outer ring
1 2 3
5 day construct 5 week construct Intact explant
Intact explant Intact explant Intact explant
4
5 day construct 5 week construct Treated explant
Treated explant Treated explant Treated explant
Fig. 2. Experimental groups. Six groups of composites were established using discs made of ( a ) 5 day constructs, (b) 5 week constructs, (c) untreated or (d) trypsin-treated explants, in conjunction with untreated or trypsin-treated explant rings; stain: safranin-0. Representative appearance of a composite shown for group 1 en face (e) and in cross-section (f) after 2 weeks of bioreactor cultivation; stain: safranin-0; scale bar: 1 mm. The table summarizes component tissues in the six groups of composites
(Fig. 2(a)). The construct periphery contained predominantly mature single chondrocytes embedded in extracellular matrix with GAG concentration that was markedly higher than in the inner tissue phase but lower than in cartilage explants; a monolayer of densely packed rounded cells was present at the surface. The inner tissue phase contained areas with immature cells starting to produce cartilaginous matrix, thereby separating themselves from the polymer fibers and each other. In contrast, 5 week constructs (Fig. 2(b)) appeared cartilaginous over their entire cross-sections. In the inner tissue phase, polymer fibers were partially degraded and immature cells were replaced by mature chondrocytes. A 30-100 pm thick layer at the construct periphery contained immature elongated cells and little GAG, and resembled the inner layer of native perichondrium. Untreated explants (Fig. 2(c)) consisted of mature chondrocytes, either single or in isogenous groups, surrounded by cartilaginous matrix with the highest concentration of GAG of the four groups. No characteristic cells are present at the explant surfaces because they were cored from the middle sections of full thickness cartilage. Empty channels correspond to blood vessels in immature cartilage. Trypsin treated explants (Fig. 2(d))consisted of an external zone containing little or no GAG, and an internal zone with GAG concentration comparable to that in untreated explants. The initial compositions are shown in Table 1 for 5 day constructs (groups 1 and 4), 5 week constructs (groups 2 and 5), intact explant rings (groups 1-3), trypsin treated explant discs and rings with 0.6 mm thick peripheral zone (group 6), and trypsin treated explant rings with 1.2 mm thick peripheral zone (groups 4-5).
Over 4 weeks of bioreactor cultivation, the wet weights of composite discs and rings increased in all groups by approximately two times (from 50 f 10 to 100 f 24 mg per disc and from 1 4 9 f 9 to 2 7 6 f 9 9 mg per ring, n = 18). GAG fraction increased in constructs and treated explants and decreased in untreated explants, and the collagen fraction increased in constructs and decreased in explants. Table 1 summarizes the biochemical compositions of composite discs and rings in all six experimental groups after 4 weeks in culture; the same trends were maintained after 8 weeks in culture (data not shown). The 5-day construct discs (groups 1 and 4) had a high cellularity, contained small amounts of GAG and collagen (Table 1) and were too fragile to allow the measurement of mechanical properties. The 5 week construct discs (groups 2 and 5) had normal cellularity, relatively higher but subnormal wet weight fractions of GAG and collagen (Table l), and an equilibrium modulus of 2 2 4 f 4 4 kPa. Untreated and treated explant discs had normal cellularity and collagen content, markedly different wet weight fractions of GAG (5.7 k 0.06 and 3.66 f 0.18, respectively (Table 1)) and equilibrium moduli of 928 f 30 kPa. After 4 weeks of composite cultivation, disc cellularities reached a plateau at a normal level, while GAG and collagen fractions remained subnormal in both 5 day and 5 week construct discs, and were at normal level in both untreated and treated explant discs. The outer rings had normal cellularity in all groups and at all time points. GAG fractions in explant rings became comparable for all groups after 4 weeks of composite cultivation (Table 1). The corresponding collagen fractions decreased in
B. Obradovic et al. I Journal of Orthopaedic Research 19 (2001) 1089-1097
1093
Table 1 Biochemical compositions of composite discs and ringsa Experimental group (see Fig. 2)
Cells (% wet weight)
GAG (% wet weight)
Collagen (YOwet weight)
Central disc
Outer ring
Central disc
Outer ring
Central disc
Outer ring
Initial composites (at the time of preparation) I 1.51 ! ~ 0 . 1 4 ~ 1 ~ 0.69f0.06 0.80 f0.13 0.69 f 0.06 2 3 0.85 f0.09 0.69 f 0.06 4 1.51 f 0.14b,d 0.68 f 0.10 5 0.80 f 0.13 0.68 f 0.10 0.75 f 0.03 6 0.98 f0.1 1
1.88 fO . l l d 2.54 f 0.09d 5.70 f0.06b 1.88f0.11 2.54 f 0.09 3.66 f0.18
6.42 i0.74 6.42 f 0.74 6.42 f0.74 1.34 i0.29h 1.34 f0.29b 3.95 f 0.60
1.10 fO.OSd 2.34 f0.35* 9.54 f 2.2b 1.10f 0.06d 2.34 f0.35d 8.93 f 0.31b
11.1 f 1.6 ll.lf1.6 11.1 f 1 . 6 11.8f1.5 11.8f 1.5 10.2f 1.3
Cultured composites (4 weeks in rotating bioreactors) 1 1.00 f 0.08'1~ 0.63 f 0.10 2 0.76 & 0.06 0.64 f0.08 3 0.65 f0.06 0.58 f 0.04 4 1.10 f 0.08' 1.00 f 0.05 5 0.96 f 0.01 1.09 f0.08 6 0.84 f0.07 0.76 f 0.06b
2.81 f 0.38 3.27 f0.18 5.17 f 0.43b 2.99 f 0.31 2.12 f 0.26 5.23 f 1.02b)d
3.83 f 0.34' 4.96 i0.70 5.82 f0.77 3.76 f.O.3lc 3.15 f 0.10" 5.65 f 0.50Czb
2.60 f 0.23d 3.06 f 0.12 5.97 f 1.lh 2.97 f 0.1Sd 3.09 f 0.17d 7.06 f 2.37b
7.61 f 0 . 2 1 5.86f0.89' 5.83 f0.77' 9 . 2 f 1.4 11.1~k3.6~ 7.18f2.1
"Statistically significant differences (P< 0.05, n = 3). ~ other ~ . groups; same time point. "4 weeks vs. initial; same group. Disc vs. ring; same group; same time point. b
rings from all composite groups except for groups 4 and 5 where it was maintained at the initial level. Integration of 5 day constructs and untreated explant rings involved the progressive formation of cartilaginous tissue at the disc-ring interface (Fig. 3). After 1 week of composite cultivation (Figs. 3(a) and (d)), the central
disc and the region at the disc-ring interface consisted of immature tissue with a high density of elongated cells, low concentration of GAG and many undegraded polymer fibers. Only a few small islands of tissue containing rounded cells surrounded by GAG indicated the maturation of the forming tissue. Empty spaces were
Fig. 3. Integration patterns. (a)-(f) Effects of composite cultivation time. Disc-ring interfaces of group 1 composites, shown in cross-section at low magnification (a)-(c) and high magnification (d)qf). Composite cultivation time: (a) and (d) 1 week, (b) and (e) 2 weeks, (c) and (f) 4 weeks. Stain: safranin 0;scale bar: (a)-(c) 1 mm or ( d H f ) 100 pn; ring on left and disc on right of each image. (g) and (h) Expression of GAG and type I collagen. Low magnification sections of disc-ring interface stained with (8) safranin-0 and (h) immunohistochemically for collagen type I; scale bar: 1 mm. PC: proliferating cells, IC: immature cartilaginous tissue, MC: mature cartilaginous tissue.
1094
B. Ohradovic et al. I Journal of Orthopaedic Research 19 (2001) 1089-1097
still clearly visible at the disc-ring interface as well as in the inner regions of the central disc. After 2 weeks of composite cultivation (Figs. 3(b) and (e)), the inner disc contained large interconnected regions of immature and maturing tissues described above, and mature cartilage characterized by round cells within clearly defined lacunae and an organized matrix rich in GAG. The presence of immature tissue within 100-200 pni of the disc-ring interface, at the outer surfaces and around empty spaces (Fig. 3(e))was associated with the actively dividing cells able to fill the gaps and create a provisional tissue matrix containing high density of proliferating cells and little GAG. After 4 weeks in culture (Figs. 3(c) and (Q),only small areas of immature tissue were present at the interface, and even those areas contained rounded cells in their lacunae and a cartilaginous matrix. The central disc consisted of mature articular cartilage containing round cells, ECM rich in GAG and only occasional polymer fibers. Between weeks 4 and 8 in culture, the composites remained largely unchanged from the light microscopic standpoint. The progressive formation of cartilaginous tissue over the course of composite cultivation was associated with the complementary expression of GAG and type I collagen as follows. The region at the disc-ring interface containing proliferating cells and the regions of immature tissue within the central disc stained strongly with type I collagen antibody, but not with safranin-0 (regions PC and IC, respectively, in Figs. 3(g) and (h)) and type I1 collagen (data not shown). In contrast, the regions of more mature cartilaginous tissue within the outer ring and central disc stained strongly with safranin-0 and type I1 collagen antibody, but did not express type I collagen, except around blood vessels in cartilage explants (region MC in Figs. 3(g) and (h)). Maturing cartilage had an intermediate behavior, staining both for GAG and type I1 collagen (but not as strongly as cartilage) and type I collagen (but not as strongly as immature tissue).
Fig. 4 shows representative histological sections of composites after 4 weeks of cultivation. As compared to the other groups, the disclring interface of group 1 composites contained a 150-200 pm thick zone of proliferative tissue with a relatively high cell density (1070 f 300 cells/mm2) and only a small amount of GAG (Fig. 4(a)). The cells appeared to be primarily rounded chondrocytes in their characteristic lacunae, with a small fraction of elongated cells. No such reparative zone was observed in composites made using 5 week constructs or cartilage explants (Figs. 4(b) and (c)), where the tissue within the central disc was cartilaginous even at the earliest time points. Gaps between the central disc and outer ring remained at the 4 week time point, and did not contain proliferating cells, but rather an acellular tissue matrix (640 f 130 and 430 90 cells/ mm2 for groups 2 and 3 composites, respectively). Trypsin treatment of cartilage explants induced chondrocytes to proliferate and form isogenous groups of 2-3 cells which rapidly produced GAG to replenish the matrix lost during the trypsin treatment. The integration patterns were markedly affected by trypsin treatment only in group 4 composites based on 5 day constructs but not in group 5 and group 6 composites based on 5 week constructs and treated explants, respectively (Fig. 4(d vs a)). In group 4 composites, the integrating area stained more intensely with safranin-0 than in the corresponding group 1 composites. In particular, as the tissue at the interface matured, cartilaginous tissue buds began to extend from the construct disc into the treated explant ring creating the appearance of an undulated interface, a phenomenon not observed in composites made using untreated explants. The measured adhesive strength of composites (Fig. 5) was consistent with the integration patterns observed for different experimental groups. The adhesive strengths of composites based on 5 week constructs, untreated or treated cartilage explants (groups 2, 3, 5, and 6 ) were in the range of 80-160 kPa. In contrast, the
Fig. 4. Integration patterns: effects of tissue composition. Representative cross-sections of integration interfaces in six groups of composites: (a) group 1, (b) group 2, (c) group 3, (d) group 4, (e) group 5 and (0 group 6 . Cultivation time: 4 weeks: stain: safranin-0; scale bar: 200 pm; disc on the top, ring on the bottom.
B. Obradouic et al. I Journal of Orthopuedic Research 19 (2001 I 1089-1097
500
*
-1
T
i6 1
2
3
4
5
6
Group Fig. 5. Adhesive strength of the integration interface. Adhesive strength of the integration interface is shown as a function of experimental group. Data represent the average iS.D. of n = 3 4 independent samples. (*) significantly different from all other experimental groups.
adhesive strengths of composites prepared using central disks made of 5 day constructs and either untreated or treated outer rings were, respectively, 254 f 66 kPa (group 1) and 384 f 63 kPa (group 4). Adhesive strength was therefore significantly higher for group 4 composites as compared to all other groups.
Discussion We report controlled studies of the formation of the tissue bond between engineered and native cartilage in tissue composites cultured for up to 8 weeks in bioreactors, and demonstrate that the integrative properties depend on the developmental stage of engineered constructs and the proteoglycan removal from the adjacent cartilage. The highest adhesive strength of the integration interface was observed for composites made from immature constructs and trypsin treated explants, and could be attributed to the formation of cartilaginous tissue by densely populated cells. Bioreactor studies of tissue composites can thus help relate the integrative properties of engineered constructs to the stage of their development. The integrative properties, evaluated by patterns of tissue remodeling and adhesive strength of the integrating interface, was generally higher for immature than for more mature tissues. Immature constructs, cultured for 5 days, had poor initial mechanical properties but integrated more rapidly than either maturing constructs cultured for 5 weeks, or cartilage explants. The integration of immature constructs with the adjacent cartilage was associated with the presence of proliferating cells and the formation of cartilaginous tissue bond (Figs. 3 and 4(a)) in contrast to the secretion of matrix components into a relatively acellular interface
1095
region of composites containing more mature constructs or cartilage explants (Figs. 4(b) and (c)). The observed integration patterns correlated with the adhesive strength of the tissue interface, which was markedly higher for immature constructs than for either mature constructs or cartilage explants (Fig. 5). This suggests that the integration patterns involving only the deposition of ECM proteins (i.e., in composites based on 5 week constructs or cartilage explants) result in lower adhesive strengths than the integration patterns which involve active tissue remodeling by proliferative cells (i.e. in composites based on 5 day constructs). Previous studies of the integrative properties of cartilage explants cultured in vitro for 3 weeks and assessed mechanically in a tensile single lap configuration [25] also reported that the adhesive strength depended on the presence on viable cells at tissue surfaces. A recent study of in vitro engineered cartilage/bone composites [28] also demonstrated that the integration at the cartilage/bone interface was better in composites consisting of immature (1 week old) than mature (4 week old) constructs. The developmental stage of the tissue determined the compositions of both the central discs and outer rings in tissue composites (Table 1, Fig. 4). Over 8 weeks of cultivation, the compositions of discs and rings approached each other in all groups, which is likely to play a role in functional response of the tissue graft to physiological loads. The associated changes in the concentrations of tissue components were higher for composites made using immature than mature tissues, a trend consistent with previously observed decreases in construct synthesis rates of GAG and collagen with cultivation time [ 1 11. Likewise, low concentrations of GAG in central discs of group 5 composites might be attributed to relatively low synthesis rates and the loss of newly synthesized GAG into the treated explant rings. These observations indicate that the compositions of the adjacent tissues depend on one another, and on developmental stage of the central discs as well as trypsin treatment of the outer rings. Partial removal of the tissue GAG can enhance cartilage remodeling. Proteoglycans are known to inhibit cell adhesion [26], and this effect can be reversed by treating cartilage explants with proteolytic enzymes [36]. Partial removal of proteoglycans by enzyme treatment of the chondral surface and local introduction of mitogenic factors were reported to improve cell recruitment from the synovial membrane, defect coverage and repair [19,20]. It has been long known that the removal of ECM induces chondrocytes to resume DNA synthesis and proliferate [1,2,18]. Under the conditions of the present study, trypsin treatment resulted in partial removal of GAG without significant change in collagen concentration. The treatment affected the integration patterns in composites made using immature (5 day) constructs (Figs. 4(a) and (b)), but had no apparent
1096
cdic Reseurch I 9 (2001) 1089-1097 B. Obradouic et af. I Journul of Orthopa$
effect on composites based on 5 week constructs or cartilage explants (Fig. 4(c)-(f)). The presence of proliferating cells was key for the progressive remodeling of the integrating interface which involved “budding” of the newly synthesized tissue into the trypsin treated explants (Fig. 4(b)). Devitalized lamb cartilage explants seeded with viable chondrocytes and implanted in nude mice produced large buds of new cartilage penetrating into the devitalized tissue [24]. Relatively small buds observed in our study were probably due to better preserved structure of trypsin treated explants as compared to devitalized tissue. The highest adhesive strength of the tissue interface of composites based on 5 day constructs and trypsin treated explants suggests that GAG removal from the adjacent tissue may further increase the integrative properties of engineered cartilage grafts. Possible mechanisms of action may involve the enhancement of cell migration and proliferation by increasing the permeability of the adjacent tissue, which is consistent with little if any effect of trypsin treatment on integration patterns of 5 week constructs and cartilage explants that did not involve the presence of proliferating cells. The progressive formation of cartilaginous tissue at the disc-ring interface involved the synthesis and ‘assembly of extracellular matrix by densely populated cells and the shift in gene expression away from type I collagen. Initially, discs made of 5 day constructs contained high density of rapidly proliferating elongated cells that resembled immature mesenchymal cells within a loose matrix that contained very little GAG and stained positively for type I collagen (Figs. 3(g)-(j)). Processes at the disc-ring interface resembled some of the embryonic events at the growth plate, where “early” cartilage produced by limb mesenchyme is also composed of predominantly type I collagen [16]. The appearance of type I1 collagen in stage 23-24 limb buds [30] coincided with the differentiation of the condensed mesenchymal cells into chondroblasts [ 161. Likewise, the progressive formation of cartilaginous matrix in the central disc and at the disc-ring interface coincided with the lack of expression of type I collagen. As a result, the majority of the tissue in the central discs of composites based on 5 day constructs and explant rings matured into well organized cartilaginous tissue matrix by 4-8 weeks in culture. In summary, bioreactors can provide controlled environment for studies of factors affecting the integration of engineered and native cartilage with the adjacent tissue without systemic effects and variability that are inherent for in vivo studies. Bioreactor studies can thus help in the planning and interpretation of complementary in vivo studies addressing the entirety of biochemical and physical regulatory signals that modulate cartilage function in an articular joint. The above findings suggest that the most important factor for inte-
grative tissue repair is the presence of biosynthetically active cells, capable of proliferating, filling the gaps at tissue interface, and progressively forming cartilaginous tissue. The duration of bioreactor cultivation for engineered constructs could be selected to achieve a desired combination of compressive stiffness (which increases with time in culture) and integrative properties (which decrease with time in culture). The use of enzymes or additional chondrogenic cells at the time of implantation may further enhance the integrative tissue repair. For any given application, specific requirements are likely to further depend on cell source, construct geometry and loading conditions, and need to be determined in coordinated in vitro and in vivo studies.
Acknowledgements The authors would like to thank Li Zeng for technical help. The research was funded by the National Aeronautics and Space Administration (NASA, Grant NAG9-836).
References [l] Abbott J, Holtzer H. The loss of phenotypic traits by differentiated cells. J Cell Biol 1966;28:473-87. [2] Abbott J, Holtzer H. The effect of 5-bromodeoxyuridine on cloned chondrocytes. PNAS 1968;59:1144-51. [3] Buckwalter JA, Mankin HJ. Articular cartilage repair and transplantation. Arthritis Rheum 1998;41:133142. [4] Buschmann MD, Gluzband YA, Grodzinsky AJ, Kimura JH, Hunziker EB. Chondrocytes in agarose culture synthesize a mechanically functional extracellular matrix. J Orthop Res 1992;10:745-58. [5] Chen FS, Frenkel SR, DiCesare PE. Repair of articular cartilage defects: Part 11. Treatment options. Am J Orthop 1999;28:88-96. [6] Farndale RW, Buttle DJ, Barrett AJ. Improved quantitation and discrimination of sulphated glycosoaminoglycans by the use of dimethylmethylene blue. Biochim Biophys Acta 1986;883:173-7. [7] Frank EH, Grodzinsky AJ. Cartilage electromechanics. I. Electrokinetic transduction and the effects of electrolyte pH and ionic strength. J Biomech 1987;20:615-27. [8] Freed LE, Vunjak-Novakovic G, Biron RJ, Eagles DB, Lesnoy DC, Barlow SK, Langer R. Biodegradable polymer scaffolds for tissue engineering. Bio/Technology 1994;12:689-93. [9] Freed LE, Vunjak-Novakovic G. Cultivation of cell-polymer tissue constructs in simulated microgravity. Biotech Bioeng 1995;46:306-13. [lo] Freed LE, Langer R, Martin I, Pelli‘s N, Vunjak-Novakovic G. Tissue engineering of cartilage in space. Proc’Natl Acad Sci USA 1997;94:13885-90. [l I] 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. [12] Freed LE, Vunjak-Novakovic G. Culture of organized cell communities. Adv Drug Del Rev 1998;33:15-30. [I31 Freed LE, Martin I, Vunjak-Novakovic G. Frontiers in tissue engineering: in vitro modulation of chondrogenesis. Clin Orth Re1 Res 1999;367S:S4&58.
B. Obradovic et al. I Journal of Orthopaedic Research 19 (2001) 1089-1097
[I41 Freed LE, Vunjak-Novakovic G. Tissue engineering of cartilage. 2nd ed. In: Bronzino J, editor. Biomedical engineering handbook. Boca Raton: CRC Press;2000, p. 1-26 [Chapter 1241. [I 51 Freed LE, Vunjak-Novakovic G. Tissue culture bioreactors: chondrogenesis as a model system. 2nd ed. In: Lanza RP, Langer R, Austin JV, editors. Principles of tissue engineering. New York: Academic Press; 2000, p. 143-56 [Chapter 131. [I61 Hall H.K. Developmental and cellular skeletal biology. New York:Academic Press; 1978, p. 134-5, 164-70. [17] Hollander AP, Heathfield TF, Webber C, Twata Y , Bourne R, Rorabeck C, Poole AR. Increased damage to type I1 collagen in osteoarthritic articular cartilage detected by a new immunoassay. J Clin Invest 1994;93:1722-32. [18] Holtzer H, Abbott J, Lash J, Holtzer S. Dedifferentiation of cartilage cells. PNAS 1960;46:153342. [19] Hunziker EB, Rosenberg LC. Repair of partial-thickness defects in articular cartilage: cell recruitment from the synovium. J Bone Joint Surg A 1996;78:721-33. [20] Hunziker EB, Kapfinger E. Removal of proteoglycans from the surface of defects in articular cartilage transiently enhances coverage by repair cells. J Bone Joint Surg B 1998;80:14450. [21] Kim YJ, Sah RL, Doong JYH, Grodzinsky AJ. Fluorometric assay of DNA in cartilage explants using Hoechst 33258. Anal Biochem 1988;174:168-76. [22] O’Driscoll S. The healing and regeneration of articular cartilage. J Bone Joint Surg A 1998;80:1795-812. [23] Paget J. Healing of cartilage. Clin Orthop 1969;64:7-8. Reprinted from Lecture XII: Healing of injuries in various tissues. In: Lectures on surgical pathology. London, p. 1-262, 1853. [24] Peretti GM, Randolph MA, Caruso EM, Rossetti F, Zaleske DJ. Bonding of cartilage matrices with cultured chondrocytes: an experimental model. J Orthop Res 1998;16:89-95. [25] Reindel ES. Ayroso AM, Chen AC, Chun DM, Schinagl RM, Sah RL. Integrative repair of articular cartilage in vitro: adhesive strength of the interface region. J Orthop Res 1995;13:751-60. [26] Rich AM, Pearlstein E, Weissmann G, Hoffstein ST. Cartilage proteoglycans inhibit fibronectin-mediated adhesion. Nature 1981:293:2%6.
1097
[27] Sah RLY, Kim IJ, Doong JYH, Grodzinsky AJ, Plaas AHK, Sandy JD. Biosynthetic response of cartilage explants to dynamic compression. J Orthop Res 1989;7:619-36. [28] Schaefer D, Martin I, Shastri P, Padera RF, Langer R, Freed LE, Vunjak-Novakovic G. In vitro generation of osteochondral composites. Biomaterials 2000;21(24):2599-606. [29] Shortkroff S, Barone L, Hsu H-P, Wrenn C, Gagne T, Chi T, et al. Healing of chondraland osteochondral defects in a canine model: the role of cultured chondrocytes in regeneration of articular cartilage. Biomaterials 1996;17:147-54. [30] von der Mark H, von der Mark K, Gay S. Study of differential collagen synthesis during the development of the chick embryo by immunofluorescence. I. Preparation of collagen type I and type I1 specific antibodies and their application to early stages of the chick embryo. Dev Biol 1976;48:23749. [31] Vunjak-Novakovic G, Obradovic B, Martin I, Bursac PM, Langer R, Freed LE. Dynamic cell seeding of polymer scaffolds for cartilage tissue engineering. Biotechnol Prog 1998;14:193-202. [32] Vunjak-Novakovic G, Martin I, Obradovic B, Treppo S, Grodzinsky AJ, Freed LE. Bioreactor cultivation conditions modulate the composition and mechanical properties of tissue-engineered cartilage. J Orthop Res 1999;17:130-8. [33] Wakitani S, Kimura T, Hirooka A, Ochi T. Yoneda M, Yasui N, Owaki H, Ono K. Repair of rabbit articular surfaces with allograft chondrocytes embedded in collagen gel. J Bone Joint Surg B 1989;71:7&80. [34] Wakitani S, Goto T, Pineda SJ, Young RG, Mansour JM, Caplan AI, Goldberg VM. Mesenchymal cell-based repair of large, fullthickness defects of articular cartilage. J Bone Joint Surg A 199%76:579-92. [35] Woessner JF. The determination of hydroxyproline in tissue and protein samples containing small proportions of this imino acid. Archiv Biochem Biophys 1961;93:440-7. [36] Yamagata M, Suzuki S, Akiyama SK, Yamada KM, Kimata K. Regulation of cell-substrate adhesion by proteoglycans immobilized on extracellular substrates. J Biol Chem 1989;264:8012-8.