Chondrocyte viability in press-fit cryopreserved osteochondral allografts

ELSEVIER

Journal of Orthopaedic Research

Journal of Orthopaedic Research 22 (2004) 781-787

www.elsevier.com/locate/orthres

Chondrocyte viability in press-fit cryopreserved osteochondral allografts Madhura D. Gole a,b, Dan Poulsen ', John M. Marzo ', Seung-Hee KO a, Israel Ziv '' Orthopurdics Section, Veteruns Adniinistrution- Western Ntw. York Heulthcure Systtw, Suite

7B.

3495 Builey Are, Buffl-rlo, N Y 14215, USA

Depurtment of' Physiology und Biophysics, Stute Uniaersirj q / Neic. York at Buffulo, 124 Shurmun Hull, Bujulo, N Y 14214, USA Orthopurdics, Millurd Fillmore Hospitul, Kuleidu Health Systan, 3 Gate3 Circle, Bgffblo, N Y 14209, USA

Received 18 July 2003; accepted 19 November 2003

Abstract The viability of chondrocytes in press-fit glycerol-preserved osteochondral allografts was compared to that in fresh autografts, after transplantation into load-bearing and non-load-bearing sites in mature sheep stifle joints. We used macroscopic grading, tonometer pen indentation testing, histology, sulfate uptake and viability as determined by confocal-microscopy to assess cartilage condition. Despite there being no statistical differences between macroscopic appearance and tonometer testing of all grafts, confocal microscopy and histology demonstrated a positive effect of load-bearing placement on cryopreserved osteochondral allografts. Allografts transplanted into load-bearing sites demonstrated superior confocal microscopy-measured chondrocyte viability (77% ? 17%1sD)than those transplanted into non-load-bearing sites (25% f 2%). Load-bearing effect was not seen in autografts (78% k ISXI), and was comparable in adjacent cartilage (830/05 9%). Similarly, load-bearing allografts demonstrated histological scoring closer to that of autografts and adjacent cartilage, all of which fared significantly better than non-load-bearing allografts. Load-bearing allografts had a greater amount of fibrocartilage than autografts or adjacent cartilage but less fibrocartilage than nonload-bearing allografts. Both autografts and allografts had non-significant increases in metabolism compared to adjacent cartilage as measured by sulfate-uptake. Load-bearing placement improved chondrocyte viability of glycerol cryopreserved osteochondral allograft following a press-fit implantation. Published by Elsevier Ltd. on behalf of orthopaedic Research Society. Kcyvords: Articular cartilage; Transplantation; Press-fit; Cryopreservation; Viability; Load-bearing

Introduction Since necrotic cartilage functioned well for S years in frozen allografts [12], it is unclear whether long-term survival of cartilage is related to the viability of the chondrocytes after transplantation and their ability to maintain the structural components of the matrix [47]. The stability of transplanted cartilage is increased by the use of a press-fit osteochondral plug (OCP) due to the fact that donor cartilage will not integrate with host * Corresponding author. Address: Orthopaedics Section, Veterans Administration-Western New York Healthcare System, Suite 7B, 3495 Bailey Ave, Buffalo, N Y 14215, USA. Tel.: +I-716-862-7898; fax: +I-716-862-7248. E-muil uddress: [email protected] (I. Ziv).

cartilage [12,14,S11. The underlying bone in cartilage defects may also be damaged [23,44] and osteochondral plug transplantation may provide structurally stronger subchondral bone to enhance overlying cartilage nutrition [34]. Autografts, used in osteochondral autograft transfer system (OATS) [4] and mosaicplasty [24,36], have improved function and reduced pain in patients with chondral lesions. However, plugs are harvested from a limited number of non-load-bearing (NLB) sites [S,36] and it is difficult to find grafts with topology that matches the load-bearing (LB) recipient site. In contrast, allografts permit the use of topology-matched grafts. Orthotopic allografts, taken from the donor area that corresponds to the site of a recipient's lesion, have properties similar to the cartilage at the lesion site [I I].

0736-0266/$ - see front matter Published by Elsevier Ltd. on behalf of orthopaedic Research Society. doi: 10.10161j.orthres.2003.11.006

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Additionally, allografts are usually harvested from younger donors with enhanced cartilage properties [8,22,25,45]. Fresh massive osteochondral allografts have been clinically effective [3,11,16], however, studies of fresh small fragment allografts [37] and a fresh osteochondral plug-allograft [ 101 also look promising. Unfortunately, the limited availability of fresh articular cartilage makes tissue banking desirable. Cryopreservation increases osteochondral allograft availability while partially retaining chondrocyte metabolic function and viability [35,41]. Although load-bearing may be required to maintain articular cartilage metabolism [30], the relationship between load-bearing and adaptation of implanted plugs is unclear. It is known that prolonged joint immobilization can cause altered chondrocyte metabolism [311, matrix degradation [27],decreased cartilage thickness [53], and inferior mechanical properties [39]. This study compares the viability of press-fit glycerol-preserved osteochondral allografts to fresh autografts, 5 and 12 months after transplantation into load-bearing and non-load-bearing sites in mature sheep stifle joints.

articular cartilage; softening and swelling of cartilage was assigned grade I ; cracks and fissuring in an area half an inch or less in diameter was assigned grade 2; similar changes in an area greater than half an inch was assigned grade 3; and, erosion of cartilage down to bone was assigned grade 4. Outerbridge cartilage scores were grouped into LB or NLB, and autografts or allografts. A tonometer pen, Tono-Pen XL (model 230635; MedtronicSolan, Jacksonville, FL, USA), was applied to the cartilage surface at the center of each graft and to adjacent articular cartilage to quantify cartilage firmness (mm Hg). Histology

The osteochondral samples were fixed in 10% unbuffered formalin for a minimum of 24 h. Samples underwent de-calcification, were embedded into paraffin, sectioned into 4 pm slices, and were processed for H&E, Masson's Trichrome and Safranin-0 stains. An Articular Cartilage Repair Histologic Scoring Scale was modified from the scale described by Schreiber et al. [49] and scorers were blind to experimental groups. Histological score was determined as the sum of the scores (max 25 points) for each of the following categories: characterization of predominant tissue as hyaline articular cartilage or fibrocartilage repair tissue (max 5), quality of Safranin-0 staining (max 3), surface (max 3) and zonal (structural) integrity (max 2), cartilage thickness (max 2), degree of cellularity (max 2), morphology of repair tissue (max 3 ) , bonding between repair and host tissue (max 2), and quality of adjacent host tissue (max 3 ) . Histological assessment was conducted on six samples to allow more cartilage material for duplicate sulfate-uptake measurements. .lcSulfrrte-uptakc

Methods After receiving approval from our institutional animal care and use committee, 12 skeletally mature sheep underwent transplantation of two osteochondral plugs in each medial femoral condyle of each stifle joint. This was done under general anesthesia and sterile open arthrotomy. Half of the 48 OCPs were fresh osteochondral autografts, harvested from the non-load-bearing anterior region of the medial femoral condyles immediately before implantation; the other 24 grafts were allograft plugs harvested from the load-bearing region of the medial femoral condyles of 4 age-matched donor sheep. Allografts were cryopreserved [35]in a 15% glycerol medium by slow-freezing and stored at -70 "C up to 72 h prior to implantation. OCPs were implanted press-fit; 6 mm plugs were placed into 5 mm defects. In the right stifle joint, the allograft was placed at a load-bearing site and the autograft at a non-load-bearing site; placement was reversed in the left stifle joint (Fig. 1). OCPs were harvested and inserted using osteochondral autograft transfer system (model AR- 1980s; Arthrex, Naples, FL, USA) tools. Sheep were permitted immediate post-operative weight bearing. At 5 and 12 months following surgery, 7 and 5 sheep were sacrificed respectively. For cartilage testing, 3 sample-plugs were centered at circumference of each osteochondral graft at the graft-host junction; these 5 mm diameter sample-plug consisted of approximately 2 mm radius of transplanted graft and 3 mm of adjacent host tissue. Macroscopic appearcmce The graft and host articular cartilage surface was examined using Outerbridge grading criteria [42]. Grade 0 was defined as normal intact

Cartilage samples were separated from subchondral bone under a high-powered surgical microscope and weighed in stoppered vials. Care was taken to minimize drying. Duplicate samples were harvested from each OCP except for six samples where the duplicate was used for histological assessment. Samples were pre-incubated in 0.5 ml of Dulbecco's Modified Eagle's medium and Ham's F-12 nutrient mixture (DMEMIF12) for 30 min at 37 "C and 7Yn CO2, then, incubated in 0.2 ml of DMEM/FI2 containing 20 uCi/ml 75Sfor 4 h at 37 "C and 7% CO2. Samples were washed once in pH 6 phosphate buffered saline (PBS) containing 5 mM ethylene diamine tetraacetic acid (EDTA) and 5 mM cysteine. The cartilage samples were digested for 16 h in 0.25 ml pH 6 PBS containing 5 mM EDTA, 5 mM cysteine, and 0.25 mginil papain. For each sample, 1 ml ethanol was added and centrifuged at I4 K rpm for 3 min. The pellet was resuspended in 1 ml ethanol and precipitated for a total of three times. The final pellet was resuspended into 0.5 ml pH 6 PBS containing 5 mM EDTA and 5 mM cysteine. Following addition of scintillation fluid, 0.1 ml of each sample solution was counted for 10 min. Duplicate sample counts were averaged and standardized for weight by calculating n-moles of "sulfate-uptake per gram of cartilage. Confocal light microscopy Osteochondral samples underwent dual-staining to distinguish viable from non-viable chondrocytes [33,41]. Osteochondral samples were sliced to a thickness of I mm and then incubated for 1 h in a calcein (acetoxymethylester) and ethidium monodimer solution [33,41]. Confocal microscopy images were processed with Confocal Assistant Version 4.02 (Todd Clark Brelje). Chondrocyte viability was calculated in a high power field using image analysis software ImageJ Version 1.27 (NIMH, Bethesda. MD, USA) as percent live cells compared to total counted.

0 Autograft donor site

0AUTOGRAFT 0 ALLOGRAFT BEARING I

Medical Fcrmoral Condyles

Fig. 1. Placement of allografts and autografts within load-bearing and non-load-bearing sites of the sheep stifle joints.

Statistical unalysi5 To accommodate for the interdependence that exists between the repeated measurements taken from the same subject (sheep), a mixed linear model approach was utilized in the analyses of resulting data. In the event that group was found to be significant, pair-wise comparisons were made between groups with a multiple comparison adjustment using Student's f-tests assuming equal variances. A nominal significance level of 0.05 was used throughout the analysis. SAS (version 8.2) was used for all analyses.

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Results Macroscopic assessment

Neither gross pathology nor effusion was observed at surgical exposure of all stifle joints. At 1 year, grafts were intact with an articular surface level with surrounding cartilage (Fig. 2). Autografts and allografts demonstrated similar Outerbridge scores as surrounding cartilage. A line of demarcation between graft and surrounding tissue was still visible at 1 year. Allografts and autografts did not demonstrate a statistically significant difference in surface appearance or in cartilage firmness, as assessed with Tono-Pen XL measurements, with regards to LB or NLB site. His tology

The subchondral bone was fully remodeled in all grafts with no discernable demarcation between graft and host tissue. However, full-thickness clefts between graft and host cartilage were still visible at 1 year after implantation in all groups. LB allografts were similar to adjacent cartilage and total autografts in regards to

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thickness, cellularity and Safranin-0 staining. However, the amount of fibrocartilage in LB allografts was greater than in adjacent cartilage and less than in NLB allografts. NLB allografts received the lowest histological scores (Fig. 3); they demonstrated an increase in the ratio of fibrocartilage to hyaline cartilage and decreases in cartilage thickness, cellularity, and degree of Safranin-0 staining compared to all other groups. Overall, allografts scored 25% lower than autografts, which appeared similar to adjacent cartilage. Differences were not analyzed statistically due to the small number of samples. j '

Sulfate-uptake

Allografts (125% k 62%SD) and autografts (157%)k 67%), regardless of LB or NLB site, had higher '5sulfateuptake than adjacent cartilage at both 5 months and 1 year (Fig. 4) after transplantation. However, differences were not statistically significant. Conjocal light microscopy

Autografts and LB allografts showed viable chondrocytes evenly distributed in intact lacunae. In contrast,

Fig. 2. Macroscopic view of osteochondral plugs I year after transplantation. A line of demarcation surrounding plugs is visible in all grafts.

Histological Grading (max score 25) Hyaline cartilage predominance 0 Safranin-0 staining

Surface integrity Structural integrity W Cartilage thickness

0 Bonding to host tissue 0 Morphology of repair tissue

Adjacent cartilage quality Cellularitv Maximum Score

NON-LB Allografts n=l

LB Allografts n=2

NON-LB Autografts n=2

LB Autografts n=l

Fig. 3. Breakdown of histology scores of samples randomly selected from groups.

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Sulfate Uptake (% Adjacent Cartilage)

I

NON-LB

MLB

OCornbined

300% 250% 200% 150% 100%

I T 4 6 Q/,

T

14X"h

50% 0%

Allografts

Autografls

Fig. 4. Sulfate uptake of autografts and allografts is higher than that of adjacent cartilage.

NLB allografts demonstrated increased populations of non-viable cells, cloning of cells within lacunae and areas of non-cellular matrix. (Fig. 5). The difference between chondrocyte viability in LB (77% k 17'%SD) and NLB autografts (78Yn k 15O%) was not statistically significant. However, viability in LB allografts (77% ? 12?4), which was comparable to that of autografts, was significantly better than NLB allografts (25%-t2%; p = 0.01). Allo-

Fig. 5. Signs of chondrocyte death and cartilage degeneration are more prominent in non-load-bearing allografts compared to load-bearing allografts or all autografts.

grafts, as a group, demonstrated less viability (56%1+ 29%) than either autografts (78% k 15%) or adjacent cartilage (83% _+ 9%) (Fig. 6).

Discussion Optimally, osteochondral plugs are placed flush with surrounding articular cartilage to restore a functional load-bearing surface [32]. Press-fit implantation minimizes the gap between non-bonded tissues which may decrease micro-motion and cartilage wear [ 171. Press-fit implantation also enhances subchondral healing [ 181 and limits bone resorption and cyst formation which can be secondary to synovial fluid penetration into the graft-host interface [2]. Although, the press-fit technique is commonly used in autograft plug transplantation, it is used rarely for allografts. However, press-fit implantation of osteochondral allografts has been researched in animal models [2,35,38,43,48]. Graft and host cartilage remain distinct in animal models despite press-fit implantation techniques [2,29,48]. In this study, a macroscopic line of demarcation and corresponding histologic full-thickness clefts between graft and host cartilage were observed in autografts and allografts, even when graft and host tissue were histologically indistinguishable. Motion and load-bearing are essential to the maintenance of articular cartilage [6,20,30] and may enhance graft viability by prompting transplanted chondrocytes to adjust their metabolic activity to the new local envi-

Viability (%Viable I Total Cells) 0 Allografts

All Grafts

Autografts

-Viability

NON-LB Grafts

of Adjacent Cartilage

LB Grafts

Fig. 6 . Load-bearing allografts have viability measurements comparable t o autografts and significantly higher than non-load-bearing allografts.

M. D. Gole et al. I Journal of Orthopaedic Research 22 (2004) 781-787

ronment. Continuous passive motion promotes cartilage healing in treatments for cartilage degeneration [27,46,50] or following periosteal grafting [40]. There is no evidence that continuous passive motion, or any other mechanical stimulation, differentially benefits cartilage in LB and NLB sites. Active motion and weight bearing on a joint provide an even larger mechanical stimulus. However, no studies examine load-bearing placement within the joint and the resulting surgical outcome or viability of transplanted grafts. Our study indicates that cryopreserved cartilage survival may be better in a LB site; the difference between LB and NLB allograft viability needs to be further investigated in a larger sample size. Motion and loading stimulate chondrocyte synthetic activity [9,28] and may induce internal tissue remodeling [7,19]. Autografts are harvested from NLB donor sites [5,36], but grafts adapt successfully to a LB environment [21,29]. In the treatment of LB cartilage lesions, allografts may provide a better match than autografts since allografts possess load-bearing properties similar to the cartilage being replaced [ l 11. Allografts may need to adapt less within a LB implantation site and may perform well due to the increased mechanical stimulus resulting in better viability and higher histological scores that the NLB counterparts. Since allografts are recovering from the stresses of cryopreservation, they may be more sensitive to the LB effect than fresh autografts. Cyclic tensile stresses as those occurring during joint motion can signal reparative stimuli while reducing the inflammatory response and resulting damage. Mechanical stresses reduce the catabolic affect of mediators such as IL-1beta through regulation of signal transduction pathways [15] and may offer allografts in LB sites increased repair activity [54] and some protection from an inflammatory response. It may be that in our smaller LB plugs, when compared to massive allografts [ 121, chondrocyte viability is higher since the smaller graft is being supported by host surrounding tissues that enhance nourishment and reduce mechanical stresses thus promoting repair activity. The process of cryopreservation influences allograft cell viability. Successful DMSO-cryopreservation of bovine (greater than 80% viability [52]) and rabbit chondrocytes (mean 91% viability [47]) has been achieved demonstrating that individual cells are capable of full recovery with retention of metabolic function. However, DMSO-cryopreserved allograft plugs measured viable cells at 50% of fresh autografts up to 1 year after transplantation; furthermore, the intermediate layers were often devoid of chondrocytes with viable cells distributed between superficial and deep zones [48]. Confocal microscopy of DMSO-cryopreserved massive allografts taken from immature animals demonstrated viability only in the superficial zones of articular cartilage [41]. A study comparing different protocols of cryopreservation found glycerol to be

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superior to DMSO in causing less stress and injury in cryopreserved osteochondral allografts; grafts were harvested 3 months after transplantation and demonstrated a diminished degree of Safranin-0 staining but overall retention of microscopic appearance [35]. The superficial layers were found to be more susceptible to the deleterious effects of cryopreservation when assessed by histology and scanning electron microscopy [35]. In our study, the viability of glycerol-preserved allografts (both LB and NLB) averaged 56% at 1 year after transplantation which is similar to preservation with DMSO [48]. Chondrocyte viability and intact structure were demonstrated through the full thickness of the allograft tissue with minimal decreases in Safranin-0 staining. When partial damage was seen, the superficial layers were most often affected which may have resulted from freeze-thaw damage or rough handling surgery. Cryopreservation has not been shown to adversely affect cartilage biomechanics [26,48] and the Tonopen XL did not elicit any indication of cartilage softening. Transplantation of fresh autograft plugs demonstrated an increase in the sulfate-uptake of grafts over control [29]. We observed similarly increased rates of 35sulfate incorporation with allografts being intermediate between autografts and normal tissue. The increase in sulfate uptake of autografts observed in our study was 50% greater when compared to adjacent cartilage but was not statistically significant. Although cartilage counts were standardized per gram, the decreased cellularity of allograft chondrocytes per gram may have resulted in the intermediate increase. The increased metabolic activity recorded in our grafts was maintained through the 5-month and 1 year time point, and possibly reflects continued chondrocyte adaptation to transplantation. In our study, confocal microscopy assessment demonstrated significant differences between groups which were not reflected in macroscopic scores, Tonopen XL testing or 3ssulfate-uptake levels. Histological scoring, although done on a limited number of samples, also indicated that it may be a good parameter of cartilage condition. Cartilage viability assessment may be the most sensitive measure, especially before mild degenerative changes have manifested macroscopically or histologically. When confocal microscopy is used in conjunction with histologic, metabolic, and biomechanical assessment, chondrocyte condition can be determined with significant reliability. Improved topographic matching [I] and a combination of allogenic matching [ 131 or immunosuppressive agents may lead to an improved outcome for allografts. Combining improvements in allograft cryopreservation with a better understanding of the effects of load bearing could result in improved chondrocyte viability and an increased rate of graft survival that is closer to the graft success seen in autografts.

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Conclusions The majority of osteochondral autografts and glycerol-preserved allografts demonstrated grossly intact structure 1 year after transplantation into sheep stifle joints. Allografts transplanted into load-bearing sites demonstrated superior viability than those transplanted into non-load-bearing sites. Glycerol cryopreservation and the press-fit insertion technique may improve outcomes when compared to techniques from similar studies. Future studies are needed to compare press-fit fresh autografts with allografts.

Acknowledgements We wish to thank the following for assistance in this study: Craig Howard for confocal microscopy, Barbara Wrobel for preparation of histological slides, Mary Taub for preparation of the sulfate-uptake protocol and Gregory Wilding for statistical analysis.

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