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SECTION II ORIGINAL ARTICLES: Research

Donor Cell Fate in Tissue Engineering for Articular Cartilage Repair

Ostrander, Roger V. MD; Goomer, Randal S. PhD; Tontz, William L. MD; Khatod, Monti MD; Harwood, Frederick L. BS; Maris, Thira M. BS; Amiel, David PhD

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Clinical Orthopaedics and Related Research: August 2001 - Volume 389 - Issue - p 228-237
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Abstract

List of Abbreviations Used: DNA deoxyribonucleic acid, BCECF-AM 2′, 7′-Bis(2-carboxyethyl)-5 (6)-carboxyfluorescein, acetoxymethylester, MMP-1 matrix metalloproteinase-1, PCR polymerase chain reaction, SRY sex determining region Y

Traumatic and degenerative lesions of articular cartilage frequently cause disability. In particular, articular cartilage has limited intrinsic healing potential. 11,20 The unsuitability of total joint replacements for young, active individuals 13,21 has provided the stimulus to search for alternative treatments in the field of biologic resurfacing of joints. One factor that may contribute to this poor healing response is the lack of chondrogenic cells available for repair. 39,48 This concept has generated increasing interest in tissue engineering and transplantation of viable chondrogenic cells to enhance cartilage repair. 2,6,9,10,14,17,18,22–24,29–32,34,38,40–43,54,55,57,58 The assumption in transplanting living cells is that they survive, replicate, and synthesize new matrix. However, many questions regarding the fate of transplanted cells remain unanswered. It is unknown whether transplanted allogenic cells remain viable and contribute to repair in the host cartilage defect with time. These questions should be answered if the time, expense, and logistics of transplanting viable cells with the hope of enhancing cartilage repair are to be justified.

Perichondrium cells have been shown to possess chondrogenic potential 1,2,14,22,23,38,43,50–52,54,60 and are used for tissue engineering. Harvesting costal perichondrium allows for the isolation of chondrogenic cells without injuring intact articular cartilage. The use of biodegradable cell carriers has gained popularity because of the technical difficulties in fixing tissue graft 16 and isolated cell suspensions 9,29 into articular cartilage defects. Porous D,D,L,L polylactic acid is a suitable cell carrier that can support a high density of viable perichondrium cells. 14,15 Polylactic acid has been used as suture material, as a surgical dressing after dental extraction, and as surgical rods, plates, and screws. 5,7,8,19,26,36,37,44

The current study was designed to address some of the questions regarding cell fate after transplantation of allogenic perichondrium cells into an experimentally created osteochondral defect. The first phase of the study looked at total cell viability within the repair tissue with time. This was achieved using an in situ double-stain technique followed by confocal microscopic analysis. 14 The second phase used PCR to quantify the percentage and absolute number of donor cells remaining in the articular cartilage repair at different intervals. The first target used was a nucleic acid sequence located on the Y chromosome of male cells, the SRY gene. The SRY gene was identified in 1990 49 and subsequently was found to be the testis-determining factor. 4 The protein product has a DNA-binding region and is thought to control the downstream transcription of genes that regulate male gonadogenesis. 12 There is a 200-bp sequence that is highly conserved among mammals. 12 The second PCR target is a sequence in the promoter region of the MMP-1 gene, 28,56 a gene present in male and female cell DNA. The MMP-1 gene is unrelated to SRY and was used as an internal reference. After transplanting male cells into a female host knee, it was possible to calculate the percentage of male donor cells in the repair tissue by quantitation of the SRY PCR product (male cells) and the MMP-1 PCR product (total cells). By determining total cell number using confocal microscopic analysis, the absolute number of donor cells in the repair tissue samples with time could be calculated.

MATERIALS AND METHODS

Primary Culture

Perichondrium was obtained from the costal cartilage of 10 adult male New Zealand White rabbits 9 to 12 months old. All procedures conformed to the guidelines of the University of California Animal Subjects Committee and the American Association for Accreditation of Laboratory Animal Care. Cells were isolated from the tissue 27 and cultured in media supplemented with 10% fetal bovine serum at 37° C. The cultures were grown to confluence in approximately 1 week.

Polylactic Acid Core Preparation and Cell Seeding

Cylindrical cores (3.7 mm diameter × 3 mm deep) were prepared from cubes of porous D,D,L,L-polylactic acid (ADD Cube, Osmed, Duluth, MN). Perichondrium cell cultures were released from tissue culture plates with 5% trypsin (Gibco, Gaithersburg, MD). Cell suspensions were generated in fresh media supplemented with 10% fetal bovine serum at a concentration of approximately 80,000 cells per μL. Single polylactic acid cores were added to centrifuge tubes containing 80 μL (6×10 6 cells) of the cell suspension. The tubes were rotated at 140 revolutions per minute for 60 minutes while kept on ice. A hemocytometer confirmed that approximately 1 million cells are seeded into each plug using this method.

Surgical Model

Fifty-three female New Zealand White rabbits 9 to 12 months old and weighing 3.5 to 4.5 kg were used. The animals were given intramuscular injections of ketamine (100 mg/kg) and xylazine (8 mg/kg), and a face mask that delivered 1% isoflurane was used. A medial parapatellar incision was made on the right knee. The medial femoral condyle was exposed. A 3.7-mm diameter × 3-mm deep defect was created as far posteriorly as possible on the condyle with a 3.7-mm surgical drill bit. A final flat-ended drill bit was used to give a flat shape to the floor of the defect at a depth of 3 mm. The male perichondrium cell and polylactic acid composite graft were press fit into the defect in each experimental knee. The joint capsule and skin were closed. The rabbits were allowed unlimited cage activity after the surgery. Intramuscular buprenorphine was administered for at least 72 hours for postoperative pain control.

Viability Assessment and Calculation of Total Cell Number

Repair tissue was evaluated for cell viability using a fluorescent in situ double-stain protocol followed by confocal microscopic analysis. 15 2′, 7′-Bis(2-carboxyethyl)-5 (6)-carboxyfluorescein, acetoxymethylester, a fluorescein derivative that is metabolized by nonspecific esterases in living cell membrane to a fluorescent product, was used to stain live cells. Propidium iodide, a cell nucleus stain that is excluded by intact living cell membrane, was used to stain dead cells. At 0, 1, 2, 3, 7, and 28 days after cell-seeded polylactic acid core implantation, repair tissue was harvested from the host knees and incubated at 37° C for 40 minutes in serum-supplemented media containing 5 μmol solutions of propidium iodide and BCECF-AM. Three samples were evaluated at each time. Animals from Day 0 were euthanized on the operating table after implantation of the polylactic acid and perichondrium constructs. The tissue was harvested immediately and stained as described. The cores were cut into three approximately 1-mm thick axial sections with a scalpel blade. The stained sections were evaluated with a Zeiss LSM510 Laser Confocal Scanning Microscope equipped with a krypton and argon laser (Carl Zeiss, Inc, Thornwood, NY). Each section was scanned for propidium iodide using an LP585 filter and the beam splitters: MBS:HFT488/568, DBS1:NFT635VIS, DBS2:mirror, DBS3: plate. The frame was scanned for BCECF-AM using a BP505–550 filter and the following beam splitters: MBS:HFT488/568, DBS1:NFT570, DBS2:mirror, DBS3: plate. Green and red images were merged using the confocal microscope image processing software.

Seven coordinates were selected randomly within each 1-mm thick section. At each of the seven coordinates, optical sections were obtained in 3-μm increments, within a volume of approximately 700 μm × 700 μm × 15 μm. The total number of live and dead cells was counted using the Image Tool Analysis Program (National Institutes of Health, Bethesda, MD), and the percent viability was calculated. The total cell number per repair tissue plug also was estimated using the images. By calculating the total volume within a 3.7-mm diameter × 3.0-mm high repair tissue plug (using the volume equation for a cylinder, πr 2 H) and the total area sampled per plug, the total cell number per sample was estimated. The total volume sampled was approximately 0.5% of the total repair tissue volume. A paired t test was used to compare the means for percent viability and the total cell number at 0, 1, 2, 3, 7, and 28 days. Statistical significance was assigned when the p value was less than 5%.

This technique was validated with the use of a hemocytometer. Control samples were generated by seeding polylactic acid cores with perichondrium cells. Before and after cell seeding, the number of cells in the cell suspension was counted using a hemocytometer. This allowed estimation of the number of cells taken up into each polylactic acid scaffold. The samples then were subjected to the total cell number calculation as described. Both methods estimated that approximately 1 million cells were seeded into each polylactic acid scaffold.

Quantitative PCR—Calculation of Donor Cell Number

Primers were designed from the highly conserved region of the rabbit SRY sequence 49,59 and from the promoter region of the rabbit MMP-1 sequence. 56 The SRY primer sequence used was 5′-TGAACGCATTCATGGTGTGGT-3′ and 5′-AGTCTTTGCGCCTCCTGGAA-3′ (Retrogen, San Diego, CA). The MMP-1 primer sequence used was 5′-GGTACCAAGAGAAAGGGAGGCAAGAC-3′ and 5′-CTCGAGCAGATCCTTCTAATGCCTGGAC-3′ (Genset, La Jolla, CA). Genomic DNA was isolated from samples using phenol and chloroform extraction aided by sonication (Sonifier 350, Branson Ultrasonic Corp, Danbury, CT) and ethanol precipitation. Extracted DNA was reconstituted in 0.5X tris-ethylenediaminetetraacetic acid buffer. Standard curves of PCR amplified SRY and MMP-1 sequences were generated using a known number of male perichondrium cells. Approximately 100 million cells were released from tissue culture plates with 5% trypsin, counted with a hemocytometer, and exposed to the same phenol and chloroform genomic DNA extraction methods as described for the samples. Serial dilutions were made from this genomic DNA corresponding to 10 6 , 10 5 , 10 4 , and 10 3 cell copies per microliter. Using the dilutions, the midpoint of the linear range of amplification was determined experimentally to be 29 PCR cycles for the SRY reaction and 26 cycles for the MMP-1 reaction. 27,46 Thus, all experiments listed in the current study pertaining to SRY were done at 29 PCR cycles, and experiments pertaining to MMP-1 were done at 26 PCR cycles.

The PCR reaction mixture contained genomic DNA, 2.5 units of Taq DNA Polymerase (Promega, Madison, WI), 600 nmol/L rabbit SRY-specific oligonucleotide primers or rabbit MMP-1 oligonucleotide primers, 100 μmol/L dNTPs (Pharmacia Biotech, Piscataway, NJ), 5 μL of MgCl 2 -free 10X PCR buffer (Promega), and 2.25 mmol/L MgCl2 (Promega) in a final volume of 50 μL. All PCR reactions were done with a GeneAmp PCR System 2400 Thermal Cycler (Perkin Elmer Cetus, Norwalk, CT). Samples were loaded directly onto the heating block at 95° C to minimize the time required for the samples to reach denaturation. An initial cycle used denaturation at 95° C for 2 minutes, annealing at 55° C for 1 minute, and extension at 72° C for 2 minutes. This was followed by 28 cycles for SRY and 25 cycles for MMP-1 of denaturation at 94° C for 1 minute, annealing at 55° C for 1 minute, and extension at 72° C for 1 minute. A final period of extension at 72° C was done for 9 minutes. 45 Fifteen microliters of the PCR reaction products were electrophoresed through 2% agarose gels stained with ethidium bromide. Photographs were taken of the gels. The photographs were scanned and quantified using the National Institutes of Health Image Analysis Software (National Institutes of Health, Bethesda, MD). Standard curves of pixel intensity versus log cell number were generated by linear regression.

Using the SRY and control primers, PCR was done on the genomic DNA isolated from the experimental samples under the same conditions that were used to amplify the standard dilutions. Fifteen microliters of the PCR reaction products were electrophoresed through a 2% agarose gel stained with ethidium bromide. The sample PCR product bands were quantified using the National Institutes of Health Image Analysis Software (National Institutes of Health) and compared with the standard curves. The SRY PCR product was used to estimate the number of male donor cells in each sample. The MMP-1 PCR product was used to estimate the number of male donor and female host cells in each sample. The percentage of donor cells per sample was calculated. Because the DNA extraction process most likely does not result in 100% yield of all DNA in each repair tissue plug, confocal microscopic analysis was used to estimate total cell number per sample. The percent donor cells values were multiplied by the total cell number values obtained with confocal microscopic analysis to estimate the total number of donor cells remaining at each time. A paired t test was used to compare the means at 0, 1, 2, 3, 7, and 28 days. Statistical significance was assigned when the p value was less than 5%.

This technique also was validated with the use of a hemocytometer as described. The number of male perichondrium cells seeded into polylactic acid cores was estimated using a hemocytometer. The same samples then were subjected to the donor cell number calculation as described. Both methods estimated that approximately 1 million male donor cells were seeded into each polylactic acid scaffold.

RESULTS

At the time the animals were euthanized, there was no evidence of acute or chronic inflammation at the operative site. All knees appeared grossly normal except for the surgically created femoral defects and repair.

Viability Assessment

The first phase of the study was done to determine the potential of implanted cells to survive the host environment. Total cell viability was determined using an in situ double-stain technique followed by confocal microscopic analysis. At all times examined, multiple green fluorescing living cells were seen interspersed with various numbers of red fluorescing dead cells (Fig 1). At 0 days, approximately 94% of the cells were viable (Fig 2). The average cell viability remained 87% or greater for all times studied, indicating that the host environment did not induce massive cell death. At 3 days, the total viability was estimated at 87%, slightly lower than at the other times. As described in Materials and Methods, each 3-mm sample plug was cut into three, 1-mm thick axial sections. The viability in the top section (closest to the synovial surface) was approximately 80% (Fig 3). The viability in the middle and deep sections increased to approximately 87% and 93%, respectively. There were no statistically significant differences in cell viability among the different times.

Fig 1.
Fig 1.:
Confocal microscopic image of a sample from Day 0. Green cells (light) stained with BCECF-AM represent living cells. Red cells (arrows) stained with propidium iodide represent dead cells. The scale included on the image is in microns.
Fig 2.
Fig 2.:
Total cell viability in repair tissue estimated using an in situ double-stain protocol and confocal microscopic analysis. Each bar represents mean percent viability ± standard error.
Fig 3.
Fig 3.:
Cell viability per section (top, middle, or deep) in repair tissue. Each bar represents mean percent viability ± standard error.

At 3, 7, and 28 days, the repair tissue was found to be more cellular. The repair tissue possessed a heterogeneous cell population, with cells of various sizes and shapes. This heterogeneity first was evident at 3 days and was even more apparent at 7 and 28 days after implantation.

Calculation of Total Cell Number

Total cell number per repair tissue plug is shown in Figure 4. Cell number increased from approximately 1 million cells at Day 0 to 1.7 million cells at Day 3. After 7 and 28 days, cell number leveled off at approximately 1.6 to 1.7 million cells. The change in total cell number was significantly different between Day 0 and Days 3, 7, and 28. There was no statistically significant difference between Day 0 and Days 1 and 2.

Fig 4.
Fig 4.:
Total cell number per repair tissue sample with time estimated using an in situ double-stain protocol and confocal microscopic analysis. Each bar represents mean total cell number ± standard error.

Quantitative PCR—Calculation of Donor Cell Number

The male specificity of the SRY sequence is seen by PCR in Figure 5. A 157-bp male-specific SRY fragment was amplified from genomic DNA extracted from male perichondrium cells. However, the same product was not obtained when PCR was attempted using genomic DNA extracted from cells of a female source. Three hundred seventy-five bp MMP-1 gene fragments were readily generated from male and female genomic DNA.

Fig 5.
Fig 5.:
Agarose gel electrophoresis with ethidium bromide staining of PCR products after amplification of genomic DNA from male and female rabbits: Lane 1: SRY gene product; Lane 2: DNA ladder; and Lane 3: MMP-1 gene product. The SRY gene product can be seen (left) in male DNA but not (right) in female DNA.

The percentage of SRY-positive donor cells in the repair tissue dropped during the span of 28 days. The composite grafts contained 100% male cells at Day 0. At 1, 2, 3, 7, and 28 days, the average donor cell percentage was estimated at 96%, 27%, 18%, 22%, and 8%, respectively, indicating that the donor cells were being diluted by host cells migrating into the plug.

To clarify the fate of these transplanted cells, the total cell number and absolute donor cell number were determined in each sample, as discussed in Materials and Methods. After Day 1, there was a decline in absolute donor cell number, with a sharp drop-off at Day 2 (Fig 6). The drop at Day 2 was statistically significant. At 0, 1, 2, 3, 7, and 28 days, the absolute donor cell number was estimated at 1.09 million, 1.26 million, 0.35 million, 0.29 million, 0.38 million, and 0.14 million, respectively. The decline implies that donor cells are migrating out of the repair or are dying. There was no significant difference in donor cell number when comparing Days 0 and 1 or when comparing Days 2, 3, 7, and 28.

Fig 6.
Fig 6.:
Absolute number of donor cells in repair tissue with time. Each bar represents mean number of donor cells ± standard error.

DISCUSSION

The results showed that viable cells were present in repair tissue after allogenic perichondrium cell transplantation in a polylactic acid scaffold at 0, 1, 2, 3, 7, and 28 days after implantation. Viability of the repair tissue was maintained during a span of 28 days at levels comparable with the high Day 0 levels. Transplantation of allogenic perichondrium cells into the host environment did not result in massive cell death. Approximately 3 days after implantation, the repair tissue had a heterogeneous appearing cell population, with cells of various sizes and shapes. This heterogeneity was even more apparent at 7 and 28 days. This most likely represents an influx of host cells from the surrounding tissue, which also possesses a heterogeneous cell population.

Male donor cells are present in repair tissue for at least 28 days after implantation, although the number of donor cells declines with time. The data showed that total cell number in the repair tissues increases with time, reaching an apparent steady state at 3 days (Fig 4). The fact that a decline in donor cell number, accompanied by an increase in total cell number, is seen indicates that the male donor cell population is diluted by a significant influx of female host cells. A decline in the percentage of donor cells is possible only with an influx of host cells. Cell death or donor cell migration alone would result in donor cell percentage calculations of 100%, although the absolute donor cell number would decline. At this time it has not been clarified which host cell types are migrating into the repair. Most of the cells are most likely coming from the subchondral bone initially. Drilling into the subchondral bone before placing the graft provides easy access for the cells to move into the scaffold. The cell population also appears heterogenous, similar to the cell population in the marrow. It is possible that other cell types, such as chondrocytes or synoviocytes, also are migrating into the repair with time. The theory of cell influx is supported by the study of Shapiro et al, 48 which examined the histologic features of surgically created knee osteochondral defects in a rabbit model. At 3 days after injury, cells were prominent at the periphery of the defect and had migrated toward the center of the clot. At 7 days, cells had infiltrated the defects extensively and had begun to synthesize a fibrous scaffold.

The decline in donor cell number was most significant between 1 and 2 days after implantation. Although it is not clear why this period is so critical, the decline in donor cell number can be explained by two possible mechanisms. The first possible mechanism is donor cell demise, by mechanisms such as immune-mediated death, apoptosis, or necrosis secondary to the trauma associated with transplantation. However, significant cell death did not appear to be present in the current model. The second possible mechanism is migration of male donor cells out of the repair and into the surrounding tissue. Maintenance of overall cell viability in the repair tissue is possible if minor donor cell death is accompanied by a larger influx of healthy female cells. Maintenance also is possible if donor cells are leaving the repair plug and entering the surrounding tissue. Based on the lack of significant cell death, the decline in donor cell number is likely attributable to the migration of male donor cells out of the repair. There is certainly some degree of donor cell death after transplantation; however, it probably is slight. Although a review of the literature has not revealed similar studies that have quantified the number of perichondrium cells with time after transplantation, Jackson et al 33 studied fresh osteochondral allografts in a goat model. Using DNA probes, they documented that during the course of 26 weeks there was a steady decline in donor DNA accompanied by a steady increase in host DNA. However, there was a subsequent increase in the average amount of donor DNA at 1 year when compared with 26 weeks, although the number of animals was small.

To the current authors’ knowledge, this is the first time the SRY gene has been used as a marker in the study of articular cartilage repair, although it has been used in other transplantation models. 3,25,35,47,53 The SRY gene is highly conserved on the Y chromosome of all mammals tested to date. 12 Thus, the SRY gene could be used as a marker in human and other mammalian models. Polymerase chain reaction amplification of two distinct genomic DNA targets, one male specific and one that is not gender specific, allowed the percentage of donor cells in repair tissue to be estimated. By estimating total cell number in the repair tissue using random sampling and confocal microscopic analysis, the absolute number of donor cells in repair tissue with time could be calculated.

Although some of the biologic features related to allogenic perichondrium cell transplantation into osteochondral defects in a rabbit model have been clarified, additional research is required to clarify whether the decline in donor cell number is significant in terms of cartilage repair. It is possible the absolute number of donor cells remaining at 28 days is sufficient to enhance repair. It also is possible that this decline represents inevitable eradication of donor cells that are not contributing to the synthesis of new cartilage matrix. Chu et al 14 reported that allogenic perichondrium and polylactic acid composite grafts transplanted into osteochondral defects of rabbit knees resulted in grossly acceptable repair in 90% of the animals after 1 year. However, the repair tissue possessed biochemical and histologic properties different from those of normal articular cartilage. 14 The use of cells transfected with growth factor gene(s) may be the next step to generate normal hyalinelike cartilage. Long periods of growth factor expression by these cells may be deleterious to the surrounding tissues. However, if allogenic donor cells are reduced in absolute numbers with time, a limited period of growth factor gene expression may be a valuable consequence. Use of the SRY gene as a marker for transplanted cells will continue to provide crucial information regarding cell fate in cartilage tissue engineering.

Acknowledgments

The authors thank J. B. Massie and K. L. Leslie for technical support.

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