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Integrative cartilage repair: adhesive strength is correlated with collagen deposition
ELSEVIER
Journal of Ort hopaedic Research Journal of Orthopaedic Research 19 (7001) 1105-11 13
ww w .elseviei-.com/locatelorthres
Integrative cartilage repair: adhesive strength is correlated with collagen deposition Michael A. DiMicco, Robert L. Sah * Depurtmrnt of' Bioengineering und Institute for Biomrdicul Engineering, Unirrrsity if' Cul~orniu,Sun Dicgo 9500 Gilmun Drive, Mud Code 0412, La follu, CA 920934412, LISA
Received 36 January 3001; accepted 8 February 2001
Abstract
Procedures to repair focal articular cartilage defects often result in poor integration between the host cartilage and the graft tissue, and this may be related to the lack of matrix deposition and the death of chondrocytes near a cut cartilage surface. The objective of this study was to determine if cartilage repair was related to deposition of newly synthesized collagen. The mechanical integration that occurred between two live adult bovine cartilage blocks cultured in partial apposition for two weeks was correlated with ['Hlproline incorporation, a measure of protein synthesis, of which more than 66% was accounted for by collagen. A similar level of mechanical integration occurred in sample pairs consisting of a live and killed cartilage block, and this adhesive strength was also correlated with ['Hlproline deposition into both the live and the killed blocks. In these samples, the ['Hlproline deposited into the killed cartilage appeared to originate from chondrocytes in the live cartilage, since live cells were not detected in the killed cartilage block by either viability staining or ["'Slsulfate incorporation. These results suggest a mechanism of integrative cartilage repair in which live chondrocytes within cartilage secrete matrix molecules that are components of a collagen network, and subsequent deposition of these molecules near the repair interface contributes to functional integration. 0 2001 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved.
Introduction The lack of healing of a cartilage laceration is a significant problem in the repair of articular cartilage defects. The goal of procedures to repair such defects is a resurfaced joint with tissue that resembles normal articular cartilage, both in function and structure. After implantation of either autogenic or allogenic osteochondral grafts [5,18,22,38] or engineered cartilage constructs [9,35] in these procedures, the bony portion of the graft often remodels and fuses with the host subchondral bone. However, the cartilaginous portion of these grafts often does not integrate with the host tissue to form a continuous, mechanically stable attachment, and this can lead t o graft failure [26]. Cartilage integration is also lacking following experimental lacerations of the articular surface in skeletally-mature animals [16]. Such lack of repair could result in abnormal stress distribution during physiological activity [3] and in the long term, degeneration [lo]. Even when *Corresponding author. Tel.: +I-858-534-0821: fax: +1-858-5346896. E-muil uddress: [email protected] (R.L. Sah).
healing appears to have occurred macroscopically, the collagen networks in the host and graft are not normal and contiguous under polarized light [14] or electron microscopy [30]. Remodeling of the collagen network has been implicated in integrative cartilage repair. The collagen network normally provides important mechanical functions in cartilage. It helps to resist applied tensile and shear stresses as well as the osmotic pressure generated by the high fixed charge density of glycosaminoglycans within the tissue [7,17]. In vitro culture of cartilage explants allows analysis of the metabolism of cartilage matrix by chondrocytes within their natural tissue environment, and thus, the study of biological mechanisms of integrative cartilage repair and collagen remodeling. In vitro, limited integration of apposing cartilage surfaces has been observed both histologically 1291 and mechanically [35] after long-term ( 1-6 weeks) incubation in medium containing serum and ascorbate. In addition, integrative repair occurs between apposing cartilage surfaces, seeded with chondrocytes or tissue-engineered cartilage and incubated subcutaneously in immunodeficient mice [23,24,31]. The formation of lysyl oxidasemediated collagen cross-links, which is inhibited by
0736-0266/01/$- see front matter Q 2001 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved. PII: SO73 6-0266( 0 1 ) 0 0 0 3 7-7
P-aminopropionitrile, appears to be a critical step in the development of adhesive strength between cartilage explants [2]. Taken together, these results suggest that the extent of integrative cartilage repair may be related to the level of deposition of newly synthesized collagen. The experimental laceration of cartilage is associated with local cellular responses [16,34]. Some of the chondrocytes near a cut surface of articular cartilage can die following laceration injury [I 3,14,16,30]. This cell death may occur either by apoptosis or necrosis [30,34]. Some of the other chondrocytes undergo limited proliferation, resulting in the formation of clones [30].However, these cells appear neither to repopulate the host cartilage at the injury site nor undergo a sustained increase in biosynthetic activity sufficient to result in functional integrative repair [14,16,30]. The extent of integration in a cartilage repair situation may also be dependent on the cell viability in the apposing tissue surfaces. I t has been suggested that integration between two apposing cartilage surfaces may occur in vivo even if one of the cartilage surfaces is dead (i.e., does not contain viable chondrocytes, [ 131). The present study investigated the hypotheses that integrative cartilage repair is related to the deposition of newly synthesized collagen, and that such a relationship exists for cartilage surfaces that are live or devitalized. Thus, using an in vitro model system, the objectives of this study were ( 1 ) to determine if the adhesive strength that develops between two live cartilage explants is correlated with the deposition of newly synthcsizcd collagen, and ( 3 )to determine whether adhesive strength would develop between live and killed cartilage blocks, and whether such repair might be associated with deposition of metabolic products by the live cartilage explant to and across the tissue-tissue interface into the killed cartilage block.
Materials and methods Materials were obtained as previously described [8,25]. Additionally, ~-[5-'H]proline was from Aniersham (Arlington Heights, IL, USA), ["S]sulfate was from DuPont NEN Research (Boston, MA. LISA), and the calcein AM-ethidium homodinier LIVWDEAD' viabilitylcytoloxicity kit was from Molecular Probes (Eugcne, O R , USA). Cu,.tilugl~c.xpkol t prepurutiun Ostcochondral fragments were harvested from the patellofemoral grooves of skeletally mature bovine animals (Haedrich's Tip Top Meats, Carlsbad. CA, USA) and processed to form 9 mm 5 inm =, 0.5 mm cartilage blocks [25]. Some cartilage blocks wcre killed by lyophilization ( Lypli-Lock: Labconco, Kansas City, MO. U S A ) for 16 h to lyse endogenous chondrocytes [8,28]. During this time, other blocks were maintained live by incubation in medium (DMEM with 20"0 fetal bovine serum (FBS). 100 pg/ml ascorbate, 0.1 mM non-essential amino acids, 10 mM HEPES, 2 mM L-glutamine, 4 mM L-proline, 100 U/ml penicillin, 100 pg/ml streptomycin, and 0.15 pg/ml amphotericin B ) prior to use in integration studies. Killed blocks were rehydrated in medium for 30 min prior to use. All incubation occurred in ;I humidified 37°C environment of 95'!/,,air/S'PO CO?. I
Three types of sample pairs, live:live, 1ive:killed. and killed:killed, were formed. Live:live samples were created by placing pairs of live cartilage blocks into custom-machined chambers, which in turn fit into 12-well tissue culture plates [XI.Each chamber maintained two blocks in a partially overlapping configuration, with a 4 mm x 5 mm interface region between the blocks over which integration could occur. A porous polysulfone platen ( I20 pm average pore size) was placed on top of the overlapping area to maintain the blocks in apposition during culture. Live:killed samples were created by placing a live cartilage block in apposition with a killed block, and ki1led:killed samples were formed from two killed cartilage blocks.
I n t q r u t i w repair und hio.syntfzt2.si.s in cornhinutions o/' lice unti killd mrtilugt' suniple puir.7 In one experiment, 1ive:live ( n = 30), 1ive:killed ( n = 2?), and killed: killed ( n = 14) samples from a total of five animals were cultured in medium for 14 days. In all cases, both blocks in a sample pair were from the same animal. Medium (2.25 ml/sample) was changed every other day. For the first 12 days of incubation, the medium was supplemented with 1 pCi/ml ['Hjproline. Then, each sample was washed with three changes of medium over 2 h to remove unincorporated radiolabel and maintained for an additional 2 days in medium without radiolabel. For the 1ive:killed samples from two of the animals, half ( n = 8 ) were prepared with the dead block on top; all other 1ive:killed samples were prepared with the live block on top. Mechanical integration of cultured samples was assessed by subjecting them to a single-lap shear test t o failure, as described previously [25]. Briefly, samples were removed from the culture chambers and placed into spring-loaded clamps on a Dyna Stat mechanical spectrometer (IMASS, Hingham, MA, USA). These clamps were displaced at 0.5 iiimimin while recording load. During testing, tissue hydration was maintained by continuous irrigation with phosphate-buffered saline (PBS). Since all specimens tested in this manner failed via separation at the interfkce between the two blocks, adhesive strength was determined as the maximum force divided by the overlap area. Displacement a t failure was taken as the point of maximum force. The energy imparted to the sample during testing (fracture energy) was calculated as the integral of the load4isplacement curve, approximated by Riernann summation. Structural stiffness was calculated as the maximum force normalized to the displacement at failure [XI. Samples that failed prior to mechanical testing were assigned adhesive strengths and fracture energies of 0 kPa and 0 J , respectively, and these samples were excluded from analysis of displacement at failure and structural stiffnesa. Following mechanical testing, the separated tissue blocks were analyzed for incorporated [3H]proline. Tissue blocks were solubilized by digestion with proteinase K (0.5 mg/ml for 16 h at 60°C). and portions of the digests were analyzed for radioactivity by liquid scintillation counting (Rack Beta 1214; Wallac-LKB, Turku, Finland). Since control studies showed that the blocks were cut to within 5%)of their target length and width, ['HI-CPM was normalized to the target block volume. Since the PBS bath solutions of the mechanical tests typically contained little of the overall radioactivity (0.7'1.0of the total counts in 1ive:live samples, 0.490 in the 1ive:killed samples), they were not analyzed further. Loculion, ,form, und source of incorporated
['Hlproline
In another set ofexperiments, 1ive:live ( n = ? I ) , 1ive:killed ( n = 21). and killed:killed ( n = 5 ) samples from a total of eight animals were cultured for 12 days in medium supplemented with 10 pCi/ml ['Hlproline. then washed, chased for 2 days, and tested mechanically as described above. Then, tissue representing the overlapping and nonoverlapping regions of 1ive:live and 1ive:killed samples were isolated and analyzed separately; here, the central 2.4 m m of the overlap region and the central 3.6 mm of the non-overlap region, and all other tissue portions, were grouped, weighed, solubilized with proteinase K, and analyzed for incorporated [3H]proline. For each region, the level of incorporated ['Hlproline was normalized to the wet weight of the tissue. Some tissue samples were also pooled and analyzed for [ZHIhydroxyproline 1331. The percentage of ['HI-radioactivity in newly rormed collagen (versus other proteins) wab calculated by multiplying the percentage of radioactivity in hydroxyproline by 1.2, which reflects
I107
the 1:I.zmolar ratio of hydroxypro1ine:proline in bovine type I1 collagen [I?]. Additionally, some 1ive:killed ( n = 12 from two animals) and killed: killed ( n = 2 from two animals) samples were cultured 14 days in medium and manually separated using forceps. Each separated block was then dissected into two sections representing the overlap and nonoverlap regions. Sections from four 1ive:killed samples were immediately incubated with calcein AM (2.7 pM) and ethidium homodimer ( 5 p M ) in PBS for 45 min before analysis of cell viability by epifluorescence microscopy. Sections from the other samples were analyzed for proteoglycan synthesis by culturing them for an additional 24 h in medium containing 5 pCi/ml ['SS]sulfate, washing six times over 1 h in PBS+ 1 mM sodium sulfate to remove unincorporated radiolabel, solubilizing, and assessing for radioactivity.
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Data in each experimental group were screened for normality (Kolmogorov-Smirnov goodness of fit test) and homoscedasticity (Scheffe-Box test) and logarithm-transformed (radioactivity) or square-root transformed (adhesive strength) where necessary to enforce these conditions [32]. The effects of treatments on the raw o r transformed data were assessed by ANOVA [33], with tissue source (animal) considered as a random effect. When ANOVA detected differences between groups, post-hoc comparisons were made by the Tukey test; alternatively, planned comparisons between experimental groups were made with Bonferroni correction of the significance criteria when a single experimental group was used for multiple comparisons. The dependence of adhesive strength on incorporated essed by single or multivariate linear regression. In correlation analyses and in studies to determine the location of deposited [3H]proline, radioactivity found in the ki1led:killed samples was defined as background, and was subtracted from both live and killed blocks in subsequent analyses. All statistical analyses were implemented with Systat 5.2 (Systat, Evanston, TL,USA), and all data are expressed as mean zk SEM.
- Fi LIVE
Fig. I . Effect of catrilage viability on ( A ) adhesive strength. and (B,C ) [?H]proline incorporation. Sample pairs (live:live, live:killed, and killed:killed) were cultured for 14 days, then tested mechanically to determine adhesive strength. Afterward, the separated cartilage blocks were solubilized and analyzed for ['HI-CPM. ' P < 0.05: P < 0.005. A*
Results
Integrahe repair and Diosynthesis in cornbinations of lizie and killed cartilage sample pairs
Integrative cartilage repair, measured mechanically, was related to the level of newly synthesized protein deposited in the tissue (Fig. 1, (A)-(C)).In 1ive:livesamples incubated for 2 weeks, the adhesive strength was 19.3 i4.2 kPa ( n = 63 from 12 animals). Also, structural stiffness was 0.40 f 0.66 N/mm ( n = 52),fracture energy was 436 f 90 yJ ( n = 5 8 ) , and the displacement at failure was 1.9 f 0.2 mm ( n = 47). In these samples, there was no significant difference in the amount of [3H]proline deposited into the top block and bottom blocks ( P = 0.55), and the level of incorporated [)H]proline was 134,OOOi 16,000 CPM per pair of blocks. Cartilage viability (live:live, live:killed, and killed: killed samples) had a significant effect on adhesive strength and biosynthesis (each P < 0.05). In the live: live samples, the development of adhesive strength was positively correlated with the amount of incorporated [3H]proline (Fig. 2, n = 30 samples from four animals). In these samples, the linear regression coefficient indicated that 37% of the observed variability in adhesive strength was attributable to variation in [3H]proline incorporation into the tissue. In kil1ed:killed samples, the
50
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0
100
200
300
400
13HI-CPM / 1000 Fig. 7 . Relationship between adhesive strength of 1ive:live cartilage samples and [3H]proline incorporation. Samples were cultured 14 days and radiolabeled with ['Hlproline before determination of adhesive strength and assessment of incorporated ['Hlradioactivity. Different symbols represent samples from different animals. Regression line r' = 0.37,n = 30.
development of adhesive strength was completely eliminated (0.0 f 0.0 kPa, n = 14 from five animals, P < 0.05 compared to 1ive:live) and the background level of in-
hf.A Dihficco, R.L. Suh I Journal of Orthopirrdic Resmrch 19 (2001) 1105-1112
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corporated ["Hlproline was correspondingly low (1.3 0.lo/u of that in live samples, P < 0.001, n = 14). Live and killed cartilage blocks were also able to integrate, and this integration also occurred in a manner that correlated with biosynthesis. In 1ive:killed samples, the resultant adhesive strength (32.2 4~5.1 kPa, n = 49 from eight animals) was not significantly different (P= 0.93) from that of 1ive:live samples (Fig. 1(A)). Fracture energy (455 f 111 pJ, n = 48), structural stiffness (0.62 f 0.81 N/mm), and displacement at failure ( 1.5 f 0.3 mm, n = 39) were also not different from live: live samples ( P = 0.96,0.26, and 0.08, respectively). The amount of [3H]proline incorporated into the live block from 1ive:killed samples (1 1 1,000 *12,000) was significantly higher than that into a single live block from a 1ive:live sample (Fig. 1(B);P < 0.05), as well as that into the apposing dead block (P< 0.005). The level of ['Hlproline incorporation into killed blocks in live, killed samples was greater than the background levels of the kil1ed:killed samples (Fig. 1(C), P < O.Ol), and equivalent to 4.0 f 0.6'1/0 of the total ['Hlradioactivity in the 1ive:killed samples. The location of the live block, on top of or underneath ( n = 5 and four samples from each of two animals, respectively) the killed block in live: killed samples, did not affect the level of [-?H]proline deposition into the live block ( P = 0.55) or the killed block ( P = 0.91). or the mechanical measures of integration. Multiple regression of adhesive strength on [3H]proline deposition into the live and killed blocks from 1ive:killed samples ( n = 22 samples from three animals) showed a trend of increasing integration with [3H]proline deposition into both the live ( P = 0.08) and killed ( P < 0.05) tissue (Fig. 3 ) . The coefficient of multiple determination (R') indicated that 44%) of the observed variability in adhesive strength was related to radiolabel deposition on either side of the interface.
*
Locution, f b m i , und svurce o j incorporated ['Hlproline during integrutire repair
The distribution of incorporated [3H]proline in the overlapping and non-overlapping regions of the live tissue blocks was analyzed further for three experimental groups, (1) the live (top) and (2) the live (bottom) blocks of 1ive:live samples (Fig. 4(A), n = 31 samples from five animals), and (3) the live blocks of the live: killed samples (Fig. 4(B), n = 31 blocks from five animals). For these live cartilage blocks, the radiolabel incorporation normalized per volume of tissue was significantly less in the overlap regions than in the nonoverlap regions for both the live (top) and live (bottom) blocks ( - 3 5 % ~ P < 0.05 and -46% P < 0.005, respectively), but not detectably different in the overlap and non-overlap regions of the live block of the live:killed samples (-l6%, P = 0.22). Direct comparison of normalized incorporation into the overlap regions
I 3H I-CPM per block 11000 Fig. 3. Relationship between adhesive strength of 1ive:killed cartilage samples and ['Hlproline incorporation. After culture and mechanical testing, the live and killed blocks from each sample were separately analyzed for ['Hlradioactivity. Different symbols represent samples from different animals. Best-fit plane is shown. Coefficient of multiple determination R' = 0.44. n = 22 samples from three animals.
showed corresponding trends, although not statistically significant, of differences between the live (top) block in the 1ive:live samples and either the live (bottom) block of the 1ive:live samples (-23%, P = 0.17) or the live block in the 1ive:killed samples (+25%, P = 0.22). Comparison of normalized incorporation into the non-overlap regions showed no difference between the live (top) and live (bottom) of the 1ive:live samples (-8% P = 0.50) and also not different between the live (top) of the live:live samples and the live block from 1ive:killed samples (-4!'0, P = 0.76). Thus, the major difference in radiolabel incorporation between live blocks of 1ive:live samples and 1ive:killed samples was the extent of decrease in the overlap region relative to the non-overlap region. In contrast, in the killed block of the 1ive:killed samples, the [3H]proline deposition was significantly higher (2.4-fold) in the overlap region than the non-overlap region ( P < 0.005). Also, in even the non-overlap region of the killed block of 1ive:killed samples, the level of ['Hlproline deposition was more than triple the background level measured in killed: killed samples. Analysis of the overlap and non-overlap regions for collagenous [3H]hydroxyproline indicated that a large portion of the [3H]proline incorporated into the cartilage tissue was in the form of collagen. In 1ive:live cartilage samples, 69 4%) of radiolabel in the overlap region, and 66 f 2 4 0 of the radiolabel in the non-overlap region, was determined to be in collagen ( n = 2 pools, each from 2 to 3 animals). Similarly high proportions ( P = 0.99) of incorporated radiolabel were determined
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Af.A . DiMicco. R.L. Suh I Journal oj' Orthiipurdic R m w c h 19 (2001i 1105-I 112
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killed blocks in ki1led:killed samples (425 & 201 CPM, n = 3). This level of incorporated [35S]sulfate was 0.5 f 0.6% of that in the live blocks (87,200f 32,300 CPM, IZ = 8. There was no significant difference ( P = 0.56) in [35S]incorporation between overlap and non-overlap regions of killed blocks from 1ive:killed samples.
Discussion
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LIVE (top)
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KILLED (bottom)
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Fig. 4. Distribution of ['Hlproline in overlap and non-overlap regions of (A) 1ive:live and (B) 1ive:killed samples. The position of cartilage blocks in the sample pair is indicated as top or bottom. Following culture and mechanical testing, tissue blocks were sectioned along their length, solubilized, and analyzed for [3H]radioactivity. normalized to tissue volume, n = 21 samples from five animals. The P-values resulting from comparison of the overlap and non-overlap regions are given as * P < 0.05; ** P < 0.005.
to be in collagen in the live blocks from 1ive:killed samples (67 f 5%) in overlap, 68 f 4% in non-overlap, n = 2 pools of samples, each from 2 to 3 animals), with slightly lower proportions ( P < 0.05) in killed blocks from these samples (52 f 5% overlap, 59 f 1% nonoverlap). The [3H]proline deposited in the killed block of live: killed samples appeared to have originated from live (i.e., metabolically active) cells in the live tissue block rather than from live cells located in the killed tissue block. The killed blocks of 1ive:killed samples, cultured for 14 days before incubation with cell viability dyes, showed only non-viable cells in both the overlap and non-overlap tissue regions, while the live blocks of these samples contained viable cells, as expected. Killed blocks of 1ive:killed samples also showed a level of [35S]sulfateincorporation (390 f 200 CPM, n = 7 that was not significantly ( P = 0.99) different from that of
The results of this study indicate that after two weeks of incubation in vitro, the extent of mechanical integration between pairs of adult bovine articular cartilage explants is related to collagen deposition and may be affected by processes involving the transport of newly formed molecules. The development of integration, measured as adhesive strength and fracture energy, was eliminated when protein synthesis was inhibited in both of the apposing cartilage explants by lyophilization treatment (Fig. 1). The presence of viable chondrocytes in only one of the two cartilage blocks in a sample pair was sufficient for integration (live:killed, Figs. 1 and 3), and in these cultures, incorporated ['Hlproline over background levels was detected in the killed cartilage. The adhesive strength that developed in both 1ive:iive (Fig. 2) and 1ive:killed (Fig. 3) samples was positively correlated with the extent of [3H]proline incorporation, with the majority of the incorporated [3H]proline being processed into collagen. In the 1ive:killed samples, radiolabeled biosynthetic products appeared to originate from chondrocytes in the live cartilage explant and be transported across the tissue-tissue interface and into the killed explant; this resulted in deposition of newly synthesized molecules in the killed cartilage, predominantly in the region directly apposed to the live cartilage (Fig. 4(B)). In 1ive:live samples, transport processes also appeared to have affected, and here possibly limited, integration, since the level of incorporated [3H]proline was lower in the overlapping region than the uncovered non-overlapping regions (Fig. 4(A)). Interestingly, the incorporation of ['Hlproline into live tissue was influenced by whether the apposing tissue was live or killed (Fig. l(B)). This effect was localized to the overlap region, where deposition of incorporated radiolabel was greater in live tissue when it was adjacent to a dead block (Fig. 4(B)) than when in apposition with a live block (Fig. 4(A)).This may be due to improved solute transport to and from the live tissue when it is facing a dead (but hydrated) cartilage block, resulting in enhanced matrix synthesis and deposition (e.g.. by viable cells in the live block near the interface). Indeed, a dead cartilage block does not consume nutrients from the medium, and it also does not produce waste products (from cell metabolism), which may inhibit biosynthetic processes in the live tissue.
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h1.A. DiMiccv, R.L. Suh I Journul of Orthopuedk Reseurcli 19 (-7001J 1105-1112
This in vitro study used adult bovine cartilage explants that were cultured in medium supplemented with serum and ascorbate, a culture system in which matrix biosynthetic activity [ 1 1,371 and integrative cartilage repair [2,25,29] have previously been characterized independently in live tissue. The inclusion of 20% FBS in the medium stimulates chondrocyte biosynthesis of proteoglycan and collagen [ 1 I]. In such explant cultures, a high percentage (62-77'34 of ['Hlproline is incorporated into collagen, as determined by analysis of [3H]hydroxyproline [27], and similarly high percentages (65-700/0) were found in the current study after prolonged (12 days) of radiolabeling. In addition, pulsechase studies indicate that both reducible bivalent and non-reducible trivalent collagen cross-links form in such explant cultures [ 11. The adhesive strength formed when pairs of explants are cultured in apposition was previously found to be -30 kPa after 2-3 weeks [2,35] and similar levels were also found under similar conditions in the current study (Fig. 1). Varying the culture duration may affect integration strength not only in a way dependent on the overall deposition of newly synthesized collagen, but also on collagen cross-linking. While the strength achieved in this culture system in vitro is unlikely to be sufficient to withstand loads in vivo [XI. the culture system does allow study of the relationship between integrative cartilage repair and biosynthetic processes. This study assessed several mechanical parameters in the single-lap test to characterize integration of cartilage surfaces. Since failure in this test can depend on a variety of factors besides the adhesive property, including the properties of the adherent blocks themselves [XI, it was of interest to compare the available parameters from the mechanical tests of the 1ive:live and 1ive:killed samples. The similarity of all parameters (adhesive strength, fracture energy, displacement at failure, and stiffness) between the 1ive:live and 1ive:killed groups suggests that the mechanics up to and including failure (and thus, adhesion) in these groups is similar. The method chosen to study integration of live and devitalized samples involved the lyophilization of cartilage explants in order to kill the endogenous cells. Although this process has the advantage of inhibiting protein synthesis without direct residual effects, this mechanism of cell lysis is not necessarily physiological. Alternatively, cycloheximide treatment can inhibit the protein synthesis pathway at the ribosome [31] and has been used to study altered chondrocyte metabolism in cartilage explant cultures [ 15,281. Indeed, treatment of 1ive:live pairs with cycloheximide during the 14 days of culture also completely inhibited protein synthesis and integration (data not shown). However, this and other chemical treatments to inhibit cell metabolism may also leave residual chemicals in the tissue that could adversely affect cells not intended to be treated (e.g., in an
apposing live cartilage explant block). Thus, the present study was focused on killed cartilage that was devitalized by lyophilization. The correlation between integration and ["Hlproline incorporation, together with previous studies showing that inhibition of collagen cross-linking reduces the development of adhesive strength [ 2 ] ,suggest that collagen synthesis, deposition, and processing is involved in integrative cartilage repair. The relatively low correlation coefficient (Figs. 2 and 3 ) may reflect the more specific dependence on molecular deposition at or near the interface site, rather than throughout the entire cartilage blocks. It may also reflect the lack of specificity of the radiolabel and the involvement of structural molecules other than collagens in the integrative repair process. Although collagen is the main contributor to the tensile properties of cartilage, molecules that interact with the collagen network and other extracellular matrix constituents of cartilage (e.g., decorin, fibromodulin, COMP, etc.) may contribute directly or indirectly to the development of adhesive strength. While the molecules involved and their interactions remain to be precisely established, it is possible that such molecules form a bridge that mechanically links existing collagen fibrils (e.g., on either side of the interface). Alternatively, the newly synthesized molecules may form an interpenetrating network that interacts with, but does not form covalent bonds with, the existing collagen networks. The demonstrated integration between a live and a killed cartilage explant has certain mechanistic implications. The development of adhesive strength (Fig. l), and the pattern of incorporation of [3H]proline, especially in the killed tissue (Fig. 4(B)), suggest that the deposition of newly formed collagen at an interface site is involved in integrative repair. A previous study using a different model system [23] found that development of mechanical adhesion between live cartilage, or constructs containing live chondrocytes, and killed tissue was coincident with the migration of cells into the dead tissue. In the present study, newly formed matrix molecules were found in dead cartilage following culture in contact with live tissue. This suggested that either these molecules were synthesized in the live tissue, and transported through the matrix, across the interface, and into the devitalized tissue or these molecules by were synthesized locally by live cells that had migrated into the dead tissue. Based on the [35S]sulfateincorporation results and the live/dead cellular staining, it seems likely that the former of these possibilities occurred. Indeed, autoradiographic studies with [3H]proline have shown that newly synthesized proteins can be dispersed throughout the interterritorial matrix, at some distance from their cellular source, after as little as 18 h of radiolabeling [36]. The long-term radiolabeling periods used in the current study may allow newly synthesized proteins to be transported over relatively long distances.
M . A . Dihfic-co, R.L. Suh I Journul
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The ability of cartilage surfaces in osteochondral fractures to integrate when pinned in apposition, but still subjected to physiological loading [ 191, suggests that augmentation of transport may contribute to integrative repair. Studies to investigate more closely the relationship between repair and the biological activities at or near the interface region, such as cell viability, proliferation, matrix metabolism, and molecular transport, may provide additional insight into the mechanisms of the repair process. The integration between live and killed cartilage also has a practical implication. This suggests that even if death of cells in host cartilage is induced when surgically debriding a cartilage defect [34], tissue implanted in a repair procedure may contain sufficient cell metabolic activity to induce integration. This may be true, for example, of transplanted chondrocytes, when positioned in a defect by being injected [6], being placed within a carrier biomaterial [35] or being placed within a tissue construct [4,20].
Acknowledgements
This work was supported by NASA, NIH, and NSF. NASA-NAGS- 1571; NIH-AR44058; NIH-AG07996; NlH-AR46555; NSF-9987353.
References [I] Ahsan T, Harwood FH, Amiel D , Sah RL. Kinetics of collagen crosshking in adult bovine articular cartilage. Trans Orthop Res SOC 2000;25:11I . [?] Ahsan T, Lottman LM, Harwood FL, Amiel D, Sah RL. Integrative cartilage repair: Inhibition by B-aminoproprionitrile. J Orthop Res 1999;17:850-7. [3] Ahsan T, Sah RL. Biomechanics of integrative cartilage repair. Osteoarthritis Cartilage 1999:7:?940. [4] Albrecht DR, Chen AC, Sah RL. Tailoring cylindrical tissueengineered cartilage constructs by targeted seeding of cells. Trans Orthop Res SOC1999;24:806. [5] Bobic V. Arthroscopic osteochondral autograft ti-ansplantation in anterior cruciate ligament reconsrruction: a preliminary clinical study. Knee Surg Sports Traumatol Arthrosc 1996;3: 2624. [6] Brittberg M, Lindahl A, Nilsson A, Ohlsson C, Isaksson 0, Peterson L. Treatment of deep cartilage defects in the knee with autologous chondrocyte transplantation. N Engl J Med 1994;331:889-95. [7] Buckwalter JA, Mankin HJ. Articular cartilage. Part I: Tissue design and chondrocyte-matrix interactions. J Bone Joint Surg 1997;79-A:600-11. [8] Chen AC, Nagrampa JP, Schinagl RM, Lottman LM, Sah RL. Chondrocyte transplantation to articular cartilage explants in vitro. J Orthop Res 1997;1S:791-807. [9]Chu CR, Dounchis JS, Yoshioka M, Sah RL. Coutts RD, Amiel D. Osteochondral repair using perichondrial cells: a one year study in rabbits. CIin Orthop 1997:340:120-9.
Orthopedic. Rrseurch I 9 (2001) 1105 I112
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[lo] Ghadially FN, Thomas I , Oryschak AF, LaLonde J-MA. Long term results of superficial defects in articular cartilage. A scanning electron microscope study. J Pathol 1977:121:213-7. [Ill Hascall VC, Handley CJ, McQuillan DJ, Hascall GR. Robinson HC. Lowther DA. The erect of serum on biosynthesis of proteoglycans by bovine articular cartilage in culture. Arch Biochem Biophys 1983;224:206-13. [12] Herbage D, Bouillet J, Bernengo J-C. Biochemical and physicochemical characterization of pepsin-solubilized type-11 collagen from bovine articular cartilage. Biochem J 1977;161:303-12. [13] Hjertquist SO, Lemperg R. Transplantation of autologous costal cartilage to an osteochondral defect on the femoral head. Histological and autoradiographical studies in adult rabbits after administration of 35s-sulphate and 3H-thymidine. Virchows Arch Pathol Anat Physiol Klin Med 1969;346:315-60. 141 Hjertquist SO, Lemperg R . Histological, autoradiographic and microchemical studies of spontaneously healing osteochondral articular defects in adult rabbits. Calcif Tiss Res 1971;s: 51-72. 151 Kimura JH, Caputo CB, Hascall VC. The effect of cycloheximide on synthesis of proteoglycans by cultured chondrocytes fi-om the swarm rat chondrosarconia. J Biol Chem 1981:256:4368-76, 161 Mankin HJ. The response of articular cartilage to mechanical injury. J Bone Joint Surg 1982;64-A:460 6. [I 71 Maroudas A. Physico-chemical properties of articular cartilage. In: Freeman MAR, editor. Adult Articular Cartilage, 2nd ed. Pitman Medical: Tunbridge Wells, England; 1979. p. ’IS-90. [I81 Matsusue Y, Yamamuro T, Hama H. Arthroscopic multiple osteochondral transplantation to the chondral defect in the knee associated with anterior cruciate ligament disruption. Arthroscopy 1993;9:318-21. [I91 Mitchell N, Shepard N. Healing of articular cartilage in intraarticular fractures in rabbits. J Bone Joint Surg 1980;63-A: 628-34. [20] Obradovic B, Martin 1, Padera R, Grodzinsky A. Freed L. Vunjak-Novakovic G. Integrative potential of tissue engineered cartilage: bioreactor studies. Trans Orthop Res SOC2000;2S: 616. [21] Obrig TG, Culp WJ, McKeehan WL, Hardesty B. The mechanism by which cycloheximide and related glutarimide antibiotics inhibit peptide synthesis on reticulocyte ribosomes. J Bid Chem 1971246:174-81. [22] Outerbridge HK. Outerbridge AR, Outerbridge RE. The use of a lateral patellar autologous graft for the repair of a large osteochondral defect in the knee. J Bone Joint Surg 1995: 77-A:65-72. [23] Peretti GM, Bonassar LJ, Caruso EM, Randolph MA, Trahan CA, Zaleske DJ. Biomechanical analysis of a chondrocyte-based repair model of articular cartilage. Tissue Eng 1999;5:317-26. [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] Sah RL, Chen AC, Chen SS, Li KW, DiMicco MA, Kurtis MS, et al. Articular cartilage repair. In: Koopman WJ, editor. Arthritis and Allied Conditions: A Textbook of Rheumatology. Philadelphia: Lippincott Williams & Wilkins, 2001. pp. 2264-78. “71 Sah RL, Doong JYH. Grodzinsky AJ, Plaas AHK, Sandy JD. Effects of compression on the loss of newly synthesized proteoglycans and proteins from cartilage explants. Arch Biochem Biophys 1991;286:20-9. [28] Sah RL, Kim YJ, Doong JH, Grodzinsky AJ. Plaas AHK, Sandy JD. Biosynthetic response of cartilage explants to dynamic compression. J Orthop Res 1989;7:619-36.
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[29] Scully SP, Joyce ME, Heydeman A, Bolander ME. Articular cartilage healing in vitro: modulation by bFGF and TGF-P 1. Trans Orthop Res SOC1991;16:385. [30] Shapiro F, Koido S, Glimcher MJ. Cell origin and differentiation in the repair of full-thickness defects of articular cartilage. J Bone Joint Surg 1993;75-A:532-53. [31] Silverman RP, Bonasser L, Passaretti D , Randolph MA, Yaremchuk MJ. Adhesion of tissue-engineered cartilate to native cartilage. Plast Reconstr Surg 2000:105:1393-8. [32] Sokal RR, Rohlf FJ. Biometry, third ed.. New York: WH Freeman: 1995. [33] Stern BD, Mechanic GL, Glimcher MJ. The resorption of bone collagen in tissue culture. Biocheni Biophys Res Commun 1963;13:13733. [34] Tew SR, Kwan AP, Hann A, Thomson BM, Archer CW. The reactions of articular cartilage to experimental wounding: role of apoptosis. Arthritis Rheum 2000;43:215-25.
[35] Wakitani S, Kimura T, Hirooka A, Ochi T, Yoneda M, Yasui N, et al. Repair of rabbit articular surfaces with allograft chondrocytes embedded in collagen gel. J Bone Joint Surg 1989:71-B: 7480. [36] Wong M, Wuethrich P, Eggli P, Hunziker E. Zone-specific cell biosynthetic activity in mature bovine articular cartilage: a new method using confocal microscopic stereology and quantitative autoradiography. J Orthop Res 1996;14: 49432. [37] Wright GC, Wei X, McDevitt CA, Lane BP, Sokoloff L. Stimulation of matrix formation in rabbit chondrocyte cultures by ascorbate. 1. Effect of ascorbate analogs and beta-aminopropionitrile. J Orthop Res 1988;6:397407. [38] Yamashita F, Sakakida I(, S u m F, Takai S. The transplantation of an autogeneic osteochondral fragment for osteochondritis dissecans of the knee. Clin Orthop 1985201: 43-50.
Journal of Ort hopaedic Research Journal of Orthopaedic Research 19 (7001) 1105-11 13
ww w .elseviei-.com/locatelorthres
Integrative cartilage repair: adhesive strength is correlated with collagen deposition Michael A. DiMicco, Robert L. Sah * Depurtmrnt of' Bioengineering und Institute for Biomrdicul Engineering, Unirrrsity if' Cul~orniu,Sun Dicgo 9500 Gilmun Drive, Mud Code 0412, La follu, CA 920934412, LISA
Received 36 January 3001; accepted 8 February 2001
Abstract
Procedures to repair focal articular cartilage defects often result in poor integration between the host cartilage and the graft tissue, and this may be related to the lack of matrix deposition and the death of chondrocytes near a cut cartilage surface. The objective of this study was to determine if cartilage repair was related to deposition of newly synthesized collagen. The mechanical integration that occurred between two live adult bovine cartilage blocks cultured in partial apposition for two weeks was correlated with ['Hlproline incorporation, a measure of protein synthesis, of which more than 66% was accounted for by collagen. A similar level of mechanical integration occurred in sample pairs consisting of a live and killed cartilage block, and this adhesive strength was also correlated with ['Hlproline deposition into both the live and the killed blocks. In these samples, the ['Hlproline deposited into the killed cartilage appeared to originate from chondrocytes in the live cartilage, since live cells were not detected in the killed cartilage block by either viability staining or ["'Slsulfate incorporation. These results suggest a mechanism of integrative cartilage repair in which live chondrocytes within cartilage secrete matrix molecules that are components of a collagen network, and subsequent deposition of these molecules near the repair interface contributes to functional integration. 0 2001 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved.
Introduction The lack of healing of a cartilage laceration is a significant problem in the repair of articular cartilage defects. The goal of procedures to repair such defects is a resurfaced joint with tissue that resembles normal articular cartilage, both in function and structure. After implantation of either autogenic or allogenic osteochondral grafts [5,18,22,38] or engineered cartilage constructs [9,35] in these procedures, the bony portion of the graft often remodels and fuses with the host subchondral bone. However, the cartilaginous portion of these grafts often does not integrate with the host tissue to form a continuous, mechanically stable attachment, and this can lead t o graft failure [26]. Cartilage integration is also lacking following experimental lacerations of the articular surface in skeletally-mature animals [16]. Such lack of repair could result in abnormal stress distribution during physiological activity [3] and in the long term, degeneration [lo]. Even when *Corresponding author. Tel.: +I-858-534-0821: fax: +1-858-5346896. E-muil uddress: [email protected] (R.L. Sah).
healing appears to have occurred macroscopically, the collagen networks in the host and graft are not normal and contiguous under polarized light [14] or electron microscopy [30]. Remodeling of the collagen network has been implicated in integrative cartilage repair. The collagen network normally provides important mechanical functions in cartilage. It helps to resist applied tensile and shear stresses as well as the osmotic pressure generated by the high fixed charge density of glycosaminoglycans within the tissue [7,17]. In vitro culture of cartilage explants allows analysis of the metabolism of cartilage matrix by chondrocytes within their natural tissue environment, and thus, the study of biological mechanisms of integrative cartilage repair and collagen remodeling. In vitro, limited integration of apposing cartilage surfaces has been observed both histologically 1291 and mechanically [35] after long-term ( 1-6 weeks) incubation in medium containing serum and ascorbate. In addition, integrative repair occurs between apposing cartilage surfaces, seeded with chondrocytes or tissue-engineered cartilage and incubated subcutaneously in immunodeficient mice [23,24,31]. The formation of lysyl oxidasemediated collagen cross-links, which is inhibited by
0736-0266/01/$- see front matter Q 2001 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved. PII: SO73 6-0266( 0 1 ) 0 0 0 3 7-7
P-aminopropionitrile, appears to be a critical step in the development of adhesive strength between cartilage explants [2]. Taken together, these results suggest that the extent of integrative cartilage repair may be related to the level of deposition of newly synthesized collagen. The experimental laceration of cartilage is associated with local cellular responses [16,34]. Some of the chondrocytes near a cut surface of articular cartilage can die following laceration injury [I 3,14,16,30]. This cell death may occur either by apoptosis or necrosis [30,34]. Some of the other chondrocytes undergo limited proliferation, resulting in the formation of clones [30].However, these cells appear neither to repopulate the host cartilage at the injury site nor undergo a sustained increase in biosynthetic activity sufficient to result in functional integrative repair [14,16,30]. The extent of integration in a cartilage repair situation may also be dependent on the cell viability in the apposing tissue surfaces. I t has been suggested that integration between two apposing cartilage surfaces may occur in vivo even if one of the cartilage surfaces is dead (i.e., does not contain viable chondrocytes, [ 131). The present study investigated the hypotheses that integrative cartilage repair is related to the deposition of newly synthesized collagen, and that such a relationship exists for cartilage surfaces that are live or devitalized. Thus, using an in vitro model system, the objectives of this study were ( 1 ) to determine if the adhesive strength that develops between two live cartilage explants is correlated with the deposition of newly synthcsizcd collagen, and ( 3 )to determine whether adhesive strength would develop between live and killed cartilage blocks, and whether such repair might be associated with deposition of metabolic products by the live cartilage explant to and across the tissue-tissue interface into the killed cartilage block.
Materials and methods Materials were obtained as previously described [8,25]. Additionally, ~-[5-'H]proline was from Aniersham (Arlington Heights, IL, USA), ["S]sulfate was from DuPont NEN Research (Boston, MA. LISA), and the calcein AM-ethidium homodinier LIVWDEAD' viabilitylcytoloxicity kit was from Molecular Probes (Eugcne, O R , USA). Cu,.tilugl~c.xpkol t prepurutiun Ostcochondral fragments were harvested from the patellofemoral grooves of skeletally mature bovine animals (Haedrich's Tip Top Meats, Carlsbad. CA, USA) and processed to form 9 mm 5 inm =, 0.5 mm cartilage blocks [25]. Some cartilage blocks wcre killed by lyophilization ( Lypli-Lock: Labconco, Kansas City, MO. U S A ) for 16 h to lyse endogenous chondrocytes [8,28]. During this time, other blocks were maintained live by incubation in medium (DMEM with 20"0 fetal bovine serum (FBS). 100 pg/ml ascorbate, 0.1 mM non-essential amino acids, 10 mM HEPES, 2 mM L-glutamine, 4 mM L-proline, 100 U/ml penicillin, 100 pg/ml streptomycin, and 0.15 pg/ml amphotericin B ) prior to use in integration studies. Killed blocks were rehydrated in medium for 30 min prior to use. All incubation occurred in ;I humidified 37°C environment of 95'!/,,air/S'PO CO?. I
Three types of sample pairs, live:live, 1ive:killed. and killed:killed, were formed. Live:live samples were created by placing pairs of live cartilage blocks into custom-machined chambers, which in turn fit into 12-well tissue culture plates [XI.Each chamber maintained two blocks in a partially overlapping configuration, with a 4 mm x 5 mm interface region between the blocks over which integration could occur. A porous polysulfone platen ( I20 pm average pore size) was placed on top of the overlapping area to maintain the blocks in apposition during culture. Live:killed samples were created by placing a live cartilage block in apposition with a killed block, and ki1led:killed samples were formed from two killed cartilage blocks.
I n t q r u t i w repair und hio.syntfzt2.si.s in cornhinutions o/' lice unti killd mrtilugt' suniple puir.7 In one experiment, 1ive:live ( n = 30), 1ive:killed ( n = 2?), and killed: killed ( n = 14) samples from a total of five animals were cultured in medium for 14 days. In all cases, both blocks in a sample pair were from the same animal. Medium (2.25 ml/sample) was changed every other day. For the first 12 days of incubation, the medium was supplemented with 1 pCi/ml ['Hjproline. Then, each sample was washed with three changes of medium over 2 h to remove unincorporated radiolabel and maintained for an additional 2 days in medium without radiolabel. For the 1ive:killed samples from two of the animals, half ( n = 8 ) were prepared with the dead block on top; all other 1ive:killed samples were prepared with the live block on top. Mechanical integration of cultured samples was assessed by subjecting them to a single-lap shear test t o failure, as described previously [25]. Briefly, samples were removed from the culture chambers and placed into spring-loaded clamps on a Dyna Stat mechanical spectrometer (IMASS, Hingham, MA, USA). These clamps were displaced at 0.5 iiimimin while recording load. During testing, tissue hydration was maintained by continuous irrigation with phosphate-buffered saline (PBS). Since all specimens tested in this manner failed via separation at the interfkce between the two blocks, adhesive strength was determined as the maximum force divided by the overlap area. Displacement a t failure was taken as the point of maximum force. The energy imparted to the sample during testing (fracture energy) was calculated as the integral of the load4isplacement curve, approximated by Riernann summation. Structural stiffness was calculated as the maximum force normalized to the displacement at failure [XI. Samples that failed prior to mechanical testing were assigned adhesive strengths and fracture energies of 0 kPa and 0 J , respectively, and these samples were excluded from analysis of displacement at failure and structural stiffnesa. Following mechanical testing, the separated tissue blocks were analyzed for incorporated [3H]proline. Tissue blocks were solubilized by digestion with proteinase K (0.5 mg/ml for 16 h at 60°C). and portions of the digests were analyzed for radioactivity by liquid scintillation counting (Rack Beta 1214; Wallac-LKB, Turku, Finland). Since control studies showed that the blocks were cut to within 5%)of their target length and width, ['HI-CPM was normalized to the target block volume. Since the PBS bath solutions of the mechanical tests typically contained little of the overall radioactivity (0.7'1.0of the total counts in 1ive:live samples, 0.490 in the 1ive:killed samples), they were not analyzed further. Loculion, ,form, und source of incorporated
['Hlproline
In another set ofexperiments, 1ive:live ( n = ? I ) , 1ive:killed ( n = 21). and killed:killed ( n = 5 ) samples from a total of eight animals were cultured for 12 days in medium supplemented with 10 pCi/ml ['Hlproline. then washed, chased for 2 days, and tested mechanically as described above. Then, tissue representing the overlapping and nonoverlapping regions of 1ive:live and 1ive:killed samples were isolated and analyzed separately; here, the central 2.4 m m of the overlap region and the central 3.6 mm of the non-overlap region, and all other tissue portions, were grouped, weighed, solubilized with proteinase K, and analyzed for incorporated [3H]proline. For each region, the level of incorporated ['Hlproline was normalized to the wet weight of the tissue. Some tissue samples were also pooled and analyzed for [ZHIhydroxyproline 1331. The percentage of ['HI-radioactivity in newly rormed collagen (versus other proteins) wab calculated by multiplying the percentage of radioactivity in hydroxyproline by 1.2, which reflects
I107
the 1:I.zmolar ratio of hydroxypro1ine:proline in bovine type I1 collagen [I?]. Additionally, some 1ive:killed ( n = 12 from two animals) and killed: killed ( n = 2 from two animals) samples were cultured 14 days in medium and manually separated using forceps. Each separated block was then dissected into two sections representing the overlap and nonoverlap regions. Sections from four 1ive:killed samples were immediately incubated with calcein AM (2.7 pM) and ethidium homodimer ( 5 p M ) in PBS for 45 min before analysis of cell viability by epifluorescence microscopy. Sections from the other samples were analyzed for proteoglycan synthesis by culturing them for an additional 24 h in medium containing 5 pCi/ml ['SS]sulfate, washing six times over 1 h in PBS+ 1 mM sodium sulfate to remove unincorporated radiolabel, solubilizing, and assessing for radioactivity.
P" 24
40
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'
A
p=0.93
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Data in each experimental group were screened for normality (Kolmogorov-Smirnov goodness of fit test) and homoscedasticity (Scheffe-Box test) and logarithm-transformed (radioactivity) or square-root transformed (adhesive strength) where necessary to enforce these conditions [32]. The effects of treatments on the raw o r transformed data were assessed by ANOVA [33], with tissue source (animal) considered as a random effect. When ANOVA detected differences between groups, post-hoc comparisons were made by the Tukey test; alternatively, planned comparisons between experimental groups were made with Bonferroni correction of the significance criteria when a single experimental group was used for multiple comparisons. The dependence of adhesive strength on incorporated essed by single or multivariate linear regression. In correlation analyses and in studies to determine the location of deposited [3H]proline, radioactivity found in the ki1led:killed samples was defined as background, and was subtracted from both live and killed blocks in subsequent analyses. All statistical analyses were implemented with Systat 5.2 (Systat, Evanston, TL,USA), and all data are expressed as mean zk SEM.
- Fi LIVE
Fig. I . Effect of catrilage viability on ( A ) adhesive strength. and (B,C ) [?H]proline incorporation. Sample pairs (live:live, live:killed, and killed:killed) were cultured for 14 days, then tested mechanically to determine adhesive strength. Afterward, the separated cartilage blocks were solubilized and analyzed for ['HI-CPM. ' P < 0.05: P < 0.005. A*
Results
Integrahe repair and Diosynthesis in cornbinations of lizie and killed cartilage sample pairs
Integrative cartilage repair, measured mechanically, was related to the level of newly synthesized protein deposited in the tissue (Fig. 1, (A)-(C)).In 1ive:livesamples incubated for 2 weeks, the adhesive strength was 19.3 i4.2 kPa ( n = 63 from 12 animals). Also, structural stiffness was 0.40 f 0.66 N/mm ( n = 52),fracture energy was 436 f 90 yJ ( n = 5 8 ) , and the displacement at failure was 1.9 f 0.2 mm ( n = 47). In these samples, there was no significant difference in the amount of [3H]proline deposited into the top block and bottom blocks ( P = 0.55), and the level of incorporated [)H]proline was 134,OOOi 16,000 CPM per pair of blocks. Cartilage viability (live:live, live:killed, and killed: killed samples) had a significant effect on adhesive strength and biosynthesis (each P < 0.05). In the live: live samples, the development of adhesive strength was positively correlated with the amount of incorporated [3H]proline (Fig. 2, n = 30 samples from four animals). In these samples, the linear regression coefficient indicated that 37% of the observed variability in adhesive strength was attributable to variation in [3H]proline incorporation into the tissue. In kil1ed:killed samples, the
50
-.
I
0
100
200
300
400
13HI-CPM / 1000 Fig. 7 . Relationship between adhesive strength of 1ive:live cartilage samples and [3H]proline incorporation. Samples were cultured 14 days and radiolabeled with ['Hlproline before determination of adhesive strength and assessment of incorporated ['Hlradioactivity. Different symbols represent samples from different animals. Regression line r' = 0.37,n = 30.
development of adhesive strength was completely eliminated (0.0 f 0.0 kPa, n = 14 from five animals, P < 0.05 compared to 1ive:live) and the background level of in-
hf.A Dihficco, R.L. Suh I Journal of Orthopirrdic Resmrch 19 (2001) 1105-1112
1108
corporated ["Hlproline was correspondingly low (1.3 0.lo/u of that in live samples, P < 0.001, n = 14). Live and killed cartilage blocks were also able to integrate, and this integration also occurred in a manner that correlated with biosynthesis. In 1ive:killed samples, the resultant adhesive strength (32.2 4~5.1 kPa, n = 49 from eight animals) was not significantly different (P= 0.93) from that of 1ive:live samples (Fig. 1(A)). Fracture energy (455 f 111 pJ, n = 48), structural stiffness (0.62 f 0.81 N/mm), and displacement at failure ( 1.5 f 0.3 mm, n = 39) were also not different from live: live samples ( P = 0.96,0.26, and 0.08, respectively). The amount of [3H]proline incorporated into the live block from 1ive:killed samples (1 1 1,000 *12,000) was significantly higher than that into a single live block from a 1ive:live sample (Fig. 1(B);P < 0.05), as well as that into the apposing dead block (P< 0.005). The level of ['Hlproline incorporation into killed blocks in live, killed samples was greater than the background levels of the kil1ed:killed samples (Fig. 1(C), P < O.Ol), and equivalent to 4.0 f 0.6'1/0 of the total ['Hlradioactivity in the 1ive:killed samples. The location of the live block, on top of or underneath ( n = 5 and four samples from each of two animals, respectively) the killed block in live: killed samples, did not affect the level of [-?H]proline deposition into the live block ( P = 0.55) or the killed block ( P = 0.91). or the mechanical measures of integration. Multiple regression of adhesive strength on [3H]proline deposition into the live and killed blocks from 1ive:killed samples ( n = 22 samples from three animals) showed a trend of increasing integration with [3H]proline deposition into both the live ( P = 0.08) and killed ( P < 0.05) tissue (Fig. 3 ) . The coefficient of multiple determination (R') indicated that 44%) of the observed variability in adhesive strength was related to radiolabel deposition on either side of the interface.
*
Locution, f b m i , und svurce o j incorporated ['Hlproline during integrutire repair
The distribution of incorporated [3H]proline in the overlapping and non-overlapping regions of the live tissue blocks was analyzed further for three experimental groups, (1) the live (top) and (2) the live (bottom) blocks of 1ive:live samples (Fig. 4(A), n = 31 samples from five animals), and (3) the live blocks of the live: killed samples (Fig. 4(B), n = 31 blocks from five animals). For these live cartilage blocks, the radiolabel incorporation normalized per volume of tissue was significantly less in the overlap regions than in the nonoverlap regions for both the live (top) and live (bottom) blocks ( - 3 5 % ~ P < 0.05 and -46% P < 0.005, respectively), but not detectably different in the overlap and non-overlap regions of the live block of the live:killed samples (-l6%, P = 0.22). Direct comparison of normalized incorporation into the overlap regions
I 3H I-CPM per block 11000 Fig. 3. Relationship between adhesive strength of 1ive:killed cartilage samples and ['Hlproline incorporation. After culture and mechanical testing, the live and killed blocks from each sample were separately analyzed for ['Hlradioactivity. Different symbols represent samples from different animals. Best-fit plane is shown. Coefficient of multiple determination R' = 0.44. n = 22 samples from three animals.
showed corresponding trends, although not statistically significant, of differences between the live (top) block in the 1ive:live samples and either the live (bottom) block of the 1ive:live samples (-23%, P = 0.17) or the live block in the 1ive:killed samples (+25%, P = 0.22). Comparison of normalized incorporation into the non-overlap regions showed no difference between the live (top) and live (bottom) of the 1ive:live samples (-8% P = 0.50) and also not different between the live (top) of the live:live samples and the live block from 1ive:killed samples (-4!'0, P = 0.76). Thus, the major difference in radiolabel incorporation between live blocks of 1ive:live samples and 1ive:killed samples was the extent of decrease in the overlap region relative to the non-overlap region. In contrast, in the killed block of the 1ive:killed samples, the [3H]proline deposition was significantly higher (2.4-fold) in the overlap region than the non-overlap region ( P < 0.005). Also, in even the non-overlap region of the killed block of 1ive:killed samples, the level of ['Hlproline deposition was more than triple the background level measured in killed: killed samples. Analysis of the overlap and non-overlap regions for collagenous [3H]hydroxyproline indicated that a large portion of the [3H]proline incorporated into the cartilage tissue was in the form of collagen. In 1ive:live cartilage samples, 69 4%) of radiolabel in the overlap region, and 66 f 2 4 0 of the radiolabel in the non-overlap region, was determined to be in collagen ( n = 2 pools, each from 2 to 3 animals). Similarly high proportions ( P = 0.99) of incorporated radiolabel were determined
*
Af.A . DiMicco. R.L. Suh I Journal oj' Orthiipurdic R m w c h 19 (2001i 1105-I 112
A
150
*
I
i
1
1 I09
killed blocks in ki1led:killed samples (425 & 201 CPM, n = 3). This level of incorporated [35S]sulfate was 0.5 f 0.6% of that in the live blocks (87,200f 32,300 CPM, IZ = 8. There was no significant difference ( P = 0.56) in [35S]incorporation between overlap and non-overlap regions of killed blocks from 1ive:killed samples.
Discussion
L Q)
P
B
150
-
'
p3922
'
I
c.
r 52,
LIVE (top)
3-
KILLED (bottom)
I
Fig. 4. Distribution of ['Hlproline in overlap and non-overlap regions of (A) 1ive:live and (B) 1ive:killed samples. The position of cartilage blocks in the sample pair is indicated as top or bottom. Following culture and mechanical testing, tissue blocks were sectioned along their length, solubilized, and analyzed for [3H]radioactivity. normalized to tissue volume, n = 21 samples from five animals. The P-values resulting from comparison of the overlap and non-overlap regions are given as * P < 0.05; ** P < 0.005.
to be in collagen in the live blocks from 1ive:killed samples (67 f 5%) in overlap, 68 f 4% in non-overlap, n = 2 pools of samples, each from 2 to 3 animals), with slightly lower proportions ( P < 0.05) in killed blocks from these samples (52 f 5% overlap, 59 f 1% nonoverlap). The [3H]proline deposited in the killed block of live: killed samples appeared to have originated from live (i.e., metabolically active) cells in the live tissue block rather than from live cells located in the killed tissue block. The killed blocks of 1ive:killed samples, cultured for 14 days before incubation with cell viability dyes, showed only non-viable cells in both the overlap and non-overlap tissue regions, while the live blocks of these samples contained viable cells, as expected. Killed blocks of 1ive:killed samples also showed a level of [35S]sulfateincorporation (390 f 200 CPM, n = 7 that was not significantly ( P = 0.99) different from that of
The results of this study indicate that after two weeks of incubation in vitro, the extent of mechanical integration between pairs of adult bovine articular cartilage explants is related to collagen deposition and may be affected by processes involving the transport of newly formed molecules. The development of integration, measured as adhesive strength and fracture energy, was eliminated when protein synthesis was inhibited in both of the apposing cartilage explants by lyophilization treatment (Fig. 1). The presence of viable chondrocytes in only one of the two cartilage blocks in a sample pair was sufficient for integration (live:killed, Figs. 1 and 3), and in these cultures, incorporated ['Hlproline over background levels was detected in the killed cartilage. The adhesive strength that developed in both 1ive:iive (Fig. 2) and 1ive:killed (Fig. 3) samples was positively correlated with the extent of [3H]proline incorporation, with the majority of the incorporated [3H]proline being processed into collagen. In the 1ive:killed samples, radiolabeled biosynthetic products appeared to originate from chondrocytes in the live cartilage explant and be transported across the tissue-tissue interface and into the killed explant; this resulted in deposition of newly synthesized molecules in the killed cartilage, predominantly in the region directly apposed to the live cartilage (Fig. 4(B)). In 1ive:live samples, transport processes also appeared to have affected, and here possibly limited, integration, since the level of incorporated [3H]proline was lower in the overlapping region than the uncovered non-overlapping regions (Fig. 4(A)). Interestingly, the incorporation of ['Hlproline into live tissue was influenced by whether the apposing tissue was live or killed (Fig. l(B)). This effect was localized to the overlap region, where deposition of incorporated radiolabel was greater in live tissue when it was adjacent to a dead block (Fig. 4(B)) than when in apposition with a live block (Fig. 4(A)).This may be due to improved solute transport to and from the live tissue when it is facing a dead (but hydrated) cartilage block, resulting in enhanced matrix synthesis and deposition (e.g.. by viable cells in the live block near the interface). Indeed, a dead cartilage block does not consume nutrients from the medium, and it also does not produce waste products (from cell metabolism), which may inhibit biosynthetic processes in the live tissue.
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This in vitro study used adult bovine cartilage explants that were cultured in medium supplemented with serum and ascorbate, a culture system in which matrix biosynthetic activity [ 1 1,371 and integrative cartilage repair [2,25,29] have previously been characterized independently in live tissue. The inclusion of 20% FBS in the medium stimulates chondrocyte biosynthesis of proteoglycan and collagen [ 1 I]. In such explant cultures, a high percentage (62-77'34 of ['Hlproline is incorporated into collagen, as determined by analysis of [3H]hydroxyproline [27], and similarly high percentages (65-700/0) were found in the current study after prolonged (12 days) of radiolabeling. In addition, pulsechase studies indicate that both reducible bivalent and non-reducible trivalent collagen cross-links form in such explant cultures [ 11. The adhesive strength formed when pairs of explants are cultured in apposition was previously found to be -30 kPa after 2-3 weeks [2,35] and similar levels were also found under similar conditions in the current study (Fig. 1). Varying the culture duration may affect integration strength not only in a way dependent on the overall deposition of newly synthesized collagen, but also on collagen cross-linking. While the strength achieved in this culture system in vitro is unlikely to be sufficient to withstand loads in vivo [XI. the culture system does allow study of the relationship between integrative cartilage repair and biosynthetic processes. This study assessed several mechanical parameters in the single-lap test to characterize integration of cartilage surfaces. Since failure in this test can depend on a variety of factors besides the adhesive property, including the properties of the adherent blocks themselves [XI, it was of interest to compare the available parameters from the mechanical tests of the 1ive:live and 1ive:killed samples. The similarity of all parameters (adhesive strength, fracture energy, displacement at failure, and stiffness) between the 1ive:live and 1ive:killed groups suggests that the mechanics up to and including failure (and thus, adhesion) in these groups is similar. The method chosen to study integration of live and devitalized samples involved the lyophilization of cartilage explants in order to kill the endogenous cells. Although this process has the advantage of inhibiting protein synthesis without direct residual effects, this mechanism of cell lysis is not necessarily physiological. Alternatively, cycloheximide treatment can inhibit the protein synthesis pathway at the ribosome [31] and has been used to study altered chondrocyte metabolism in cartilage explant cultures [ 15,281. Indeed, treatment of 1ive:live pairs with cycloheximide during the 14 days of culture also completely inhibited protein synthesis and integration (data not shown). However, this and other chemical treatments to inhibit cell metabolism may also leave residual chemicals in the tissue that could adversely affect cells not intended to be treated (e.g., in an
apposing live cartilage explant block). Thus, the present study was focused on killed cartilage that was devitalized by lyophilization. The correlation between integration and ["Hlproline incorporation, together with previous studies showing that inhibition of collagen cross-linking reduces the development of adhesive strength [ 2 ] ,suggest that collagen synthesis, deposition, and processing is involved in integrative cartilage repair. The relatively low correlation coefficient (Figs. 2 and 3 ) may reflect the more specific dependence on molecular deposition at or near the interface site, rather than throughout the entire cartilage blocks. It may also reflect the lack of specificity of the radiolabel and the involvement of structural molecules other than collagens in the integrative repair process. Although collagen is the main contributor to the tensile properties of cartilage, molecules that interact with the collagen network and other extracellular matrix constituents of cartilage (e.g., decorin, fibromodulin, COMP, etc.) may contribute directly or indirectly to the development of adhesive strength. While the molecules involved and their interactions remain to be precisely established, it is possible that such molecules form a bridge that mechanically links existing collagen fibrils (e.g., on either side of the interface). Alternatively, the newly synthesized molecules may form an interpenetrating network that interacts with, but does not form covalent bonds with, the existing collagen networks. The demonstrated integration between a live and a killed cartilage explant has certain mechanistic implications. The development of adhesive strength (Fig. l), and the pattern of incorporation of [3H]proline, especially in the killed tissue (Fig. 4(B)), suggest that the deposition of newly formed collagen at an interface site is involved in integrative repair. A previous study using a different model system [23] found that development of mechanical adhesion between live cartilage, or constructs containing live chondrocytes, and killed tissue was coincident with the migration of cells into the dead tissue. In the present study, newly formed matrix molecules were found in dead cartilage following culture in contact with live tissue. This suggested that either these molecules were synthesized in the live tissue, and transported through the matrix, across the interface, and into the devitalized tissue or these molecules by were synthesized locally by live cells that had migrated into the dead tissue. Based on the [35S]sulfateincorporation results and the live/dead cellular staining, it seems likely that the former of these possibilities occurred. Indeed, autoradiographic studies with [3H]proline have shown that newly synthesized proteins can be dispersed throughout the interterritorial matrix, at some distance from their cellular source, after as little as 18 h of radiolabeling [36]. The long-term radiolabeling periods used in the current study may allow newly synthesized proteins to be transported over relatively long distances.
M . A . Dihfic-co, R.L. Suh I Journul
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The ability of cartilage surfaces in osteochondral fractures to integrate when pinned in apposition, but still subjected to physiological loading [ 191, suggests that augmentation of transport may contribute to integrative repair. Studies to investigate more closely the relationship between repair and the biological activities at or near the interface region, such as cell viability, proliferation, matrix metabolism, and molecular transport, may provide additional insight into the mechanisms of the repair process. The integration between live and killed cartilage also has a practical implication. This suggests that even if death of cells in host cartilage is induced when surgically debriding a cartilage defect [34], tissue implanted in a repair procedure may contain sufficient cell metabolic activity to induce integration. This may be true, for example, of transplanted chondrocytes, when positioned in a defect by being injected [6], being placed within a carrier biomaterial [35] or being placed within a tissue construct [4,20].
Acknowledgements
This work was supported by NASA, NIH, and NSF. NASA-NAGS- 1571; NIH-AR44058; NIH-AG07996; NlH-AR46555; NSF-9987353.
References [I] Ahsan T, Harwood FH, Amiel D , Sah RL. Kinetics of collagen crosshking in adult bovine articular cartilage. Trans Orthop Res SOC 2000;25:11I . [?] Ahsan T, Lottman LM, Harwood FL, Amiel D, Sah RL. Integrative cartilage repair: Inhibition by B-aminoproprionitrile. J Orthop Res 1999;17:850-7. [3] Ahsan T, Sah RL. Biomechanics of integrative cartilage repair. Osteoarthritis Cartilage 1999:7:?940. [4] Albrecht DR, Chen AC, Sah RL. Tailoring cylindrical tissueengineered cartilage constructs by targeted seeding of cells. Trans Orthop Res SOC1999;24:806. [5] Bobic V. Arthroscopic osteochondral autograft ti-ansplantation in anterior cruciate ligament reconsrruction: a preliminary clinical study. Knee Surg Sports Traumatol Arthrosc 1996;3: 2624. [6] Brittberg M, Lindahl A, Nilsson A, Ohlsson C, Isaksson 0, Peterson L. Treatment of deep cartilage defects in the knee with autologous chondrocyte transplantation. N Engl J Med 1994;331:889-95. [7] Buckwalter JA, Mankin HJ. Articular cartilage. Part I: Tissue design and chondrocyte-matrix interactions. J Bone Joint Surg 1997;79-A:600-11. [8] Chen AC, Nagrampa JP, Schinagl RM, Lottman LM, Sah RL. Chondrocyte transplantation to articular cartilage explants in vitro. J Orthop Res 1997;1S:791-807. [9]Chu CR, Dounchis JS, Yoshioka M, Sah RL. Coutts RD, Amiel D. Osteochondral repair using perichondrial cells: a one year study in rabbits. CIin Orthop 1997:340:120-9.
Orthopedic. Rrseurch I 9 (2001) 1105 I112
1111
[lo] Ghadially FN, Thomas I , Oryschak AF, LaLonde J-MA. Long term results of superficial defects in articular cartilage. A scanning electron microscope study. J Pathol 1977:121:213-7. [Ill Hascall VC, Handley CJ, McQuillan DJ, Hascall GR. Robinson HC. Lowther DA. The erect of serum on biosynthesis of proteoglycans by bovine articular cartilage in culture. Arch Biochem Biophys 1983;224:206-13. [12] Herbage D, Bouillet J, Bernengo J-C. Biochemical and physicochemical characterization of pepsin-solubilized type-11 collagen from bovine articular cartilage. Biochem J 1977;161:303-12. [13] Hjertquist SO, Lemperg R. Transplantation of autologous costal cartilage to an osteochondral defect on the femoral head. Histological and autoradiographical studies in adult rabbits after administration of 35s-sulphate and 3H-thymidine. Virchows Arch Pathol Anat Physiol Klin Med 1969;346:315-60. 141 Hjertquist SO, Lemperg R . Histological, autoradiographic and microchemical studies of spontaneously healing osteochondral articular defects in adult rabbits. Calcif Tiss Res 1971;s: 51-72. 151 Kimura JH, Caputo CB, Hascall VC. The effect of cycloheximide on synthesis of proteoglycans by cultured chondrocytes fi-om the swarm rat chondrosarconia. J Biol Chem 1981:256:4368-76, 161 Mankin HJ. The response of articular cartilage to mechanical injury. J Bone Joint Surg 1982;64-A:460 6. [I 71 Maroudas A. Physico-chemical properties of articular cartilage. In: Freeman MAR, editor. Adult Articular Cartilage, 2nd ed. Pitman Medical: Tunbridge Wells, England; 1979. p. ’IS-90. [I81 Matsusue Y, Yamamuro T, Hama H. Arthroscopic multiple osteochondral transplantation to the chondral defect in the knee associated with anterior cruciate ligament disruption. Arthroscopy 1993;9:318-21. [I91 Mitchell N, Shepard N. Healing of articular cartilage in intraarticular fractures in rabbits. J Bone Joint Surg 1980;63-A: 628-34. [20] Obradovic B, Martin 1, Padera R, Grodzinsky A. Freed L. Vunjak-Novakovic G. Integrative potential of tissue engineered cartilage: bioreactor studies. Trans Orthop Res SOC2000;2S: 616. [21] Obrig TG, Culp WJ, McKeehan WL, Hardesty B. The mechanism by which cycloheximide and related glutarimide antibiotics inhibit peptide synthesis on reticulocyte ribosomes. J Bid Chem 1971246:174-81. [22] Outerbridge HK. Outerbridge AR, Outerbridge RE. The use of a lateral patellar autologous graft for the repair of a large osteochondral defect in the knee. J Bone Joint Surg 1995: 77-A:65-72. [23] Peretti GM, Bonassar LJ, Caruso EM, Randolph MA, Trahan CA, Zaleske DJ. Biomechanical analysis of a chondrocyte-based repair model of articular cartilage. Tissue Eng 1999;5:317-26. [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] Sah RL, Chen AC, Chen SS, Li KW, DiMicco MA, Kurtis MS, et al. Articular cartilage repair. In: Koopman WJ, editor. Arthritis and Allied Conditions: A Textbook of Rheumatology. Philadelphia: Lippincott Williams & Wilkins, 2001. pp. 2264-78. “71 Sah RL, Doong JYH. Grodzinsky AJ, Plaas AHK, Sandy JD. Effects of compression on the loss of newly synthesized proteoglycans and proteins from cartilage explants. Arch Biochem Biophys 1991;286:20-9. [28] Sah RL, Kim YJ, Doong JH, Grodzinsky AJ. Plaas AHK, Sandy JD. Biosynthetic response of cartilage explants to dynamic compression. J Orthop Res 1989;7:619-36.
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[29] Scully SP, Joyce ME, Heydeman A, Bolander ME. Articular cartilage healing in vitro: modulation by bFGF and TGF-P 1. Trans Orthop Res SOC1991;16:385. [30] Shapiro F, Koido S, Glimcher MJ. Cell origin and differentiation in the repair of full-thickness defects of articular cartilage. J Bone Joint Surg 1993;75-A:532-53. [31] Silverman RP, Bonasser L, Passaretti D , Randolph MA, Yaremchuk MJ. Adhesion of tissue-engineered cartilate to native cartilage. Plast Reconstr Surg 2000:105:1393-8. [32] Sokal RR, Rohlf FJ. Biometry, third ed.. New York: WH Freeman: 1995. [33] Stern BD, Mechanic GL, Glimcher MJ. The resorption of bone collagen in tissue culture. Biocheni Biophys Res Commun 1963;13:13733. [34] Tew SR, Kwan AP, Hann A, Thomson BM, Archer CW. The reactions of articular cartilage to experimental wounding: role of apoptosis. Arthritis Rheum 2000;43:215-25.
[35] Wakitani S, Kimura T, Hirooka A, Ochi T, Yoneda M, Yasui N, et al. Repair of rabbit articular surfaces with allograft chondrocytes embedded in collagen gel. J Bone Joint Surg 1989:71-B: 7480. [36] Wong M, Wuethrich P, Eggli P, Hunziker E. Zone-specific cell biosynthetic activity in mature bovine articular cartilage: a new method using confocal microscopic stereology and quantitative autoradiography. J Orthop Res 1996;14: 49432. [37] Wright GC, Wei X, McDevitt CA, Lane BP, Sokoloff L. Stimulation of matrix formation in rabbit chondrocyte cultures by ascorbate. 1. Effect of ascorbate analogs and beta-aminopropionitrile. J Orthop Res 1988;6:397407. [38] Yamashita F, Sakakida I(, S u m F, Takai S. The transplantation of an autogeneic osteochondral fragment for osteochondritis dissecans of the knee. Clin Orthop 1985201: 43-50.