Fresh osteochondral allografts have been widely used to treat cartilage lesions for more than 100 years1. Transplantation of cartilage and bone in the form of an allograft allows osseous healing while maintaining the articular cartilage architecture. This composite tissue transplant remains intact in vivo for extensive periods of time with a favorable mechanical and biological environment. The chondrocytes of the graft are thought to actively remodel the extracellular matrix environment, and thus contribute to the tissue integrity. We recently reported that allograft cells could survive up to twenty-nine years after transplantation without the need for systemic immunosuppression2. Although mosaic cell populations have been demonstrated in other forms of transplantation, these have always been under the umbrella of long-term systemic immunosuppression3,4. In a classic study, Langer and Gross5 showed that intact articular cartilage surfaces obtained by removing the subchondral bone of rat femoral heads and filling of the osseous segments with acrylic cement exhibited essentially no humoral immune response in contrast to that seen with minced cartilage or isolated chondrocyte transplants. This finding has been attributed to the so-called “immunoprivileged” status of articular cartilage, which protects the chondrocytes from the immune system of the host.
The extent to which allograft chondrocytes retain their gene expression profiles and chondrogenic capacities remains unknown. Our goal was to compare gene expression, proliferation rate, and chondrogenic potential between host and allograft chondrocytes isolated three years after an unsuccessful fresh osteochondral allograft transplant in the knee. The patient was informed that data concerning her case would be submitted for publication, and she provided consent. The study was performed in full compliance with our institutional review board (IRB). Our IRB at University of California Davis Medical Center does not consider case reports to be research and does not expressly provide IRB approval. We have sought IRB approval for case reports on a number of occasions in the past and have been given written documentation of this policy.
Tissue Source
A forty-eight-year-old woman with early osteoarthritis of the knee was treated with a fresh osteochondral allograft with use of cylindrical plugs prepared with commercially available instrumentation (Arthrex, Naples, Florida); the plugs were applied to the trochlea (20 × 20-mm plug), the medial femoral condyle (20 × 20-mm plug), and the lateral femoral condyle (15 × 15-mm plug). The graft was a whole fresh distal part of a femur (University of Miami Tissue Bank, Miami, Florida) from an eighteen-year-old male donor implanted twelve days after harvest. The tissue bank provided the allografts submerged in culture media (RPMI-1640 or lactated Ringer solution) with antibiotics at a refrigerated temperature (1°C to 10°C, never frozen). Transplants approximately 1.0 to 1.5 cm in diameter were created from the region of the allograft joint corresponding to the site of transplantation. Prior to implantation, bone marrow was removed from the transplant plugs with pulsatile irrigation, and the transplant was then plugged into the recipient site without any additional treatment.
The knee pain and osteoarthritis ultimately progressed, and a total knee arthroplasty was performed three years after the index procedure. Discarded tissues from the total knee arthroplasty were removed from the operative field, placed in sterile saline solution in the operating room, and processed for chondrocyte isolation within sixty minutes after surgery.
Cell Sex Determination by FISH to X and Y Chromosomal DNA
Fixed cells from cultured articular cartilage tissue were placed onto glass slides and dried, and dual-color fluorescence in situ hybridization (FISH) was performed on the cells with the chromosome enumeration probes (CEPs) X SpectrumGreen and Y SpectrumOrange (Vysis, Des Plaines, Illinois). These probes are specific to the centromere region of Xp11.1-q11.1 and Yp11.1-q11.1 of the X and Y chromosomes. Female cells (XX) demonstrate two green signals (GG); male cells (XY) demonstrate one orange and one green signal (OG). The cells were denatured and hybridized overnight with use of a HYBrite system (Vysis). The slides were washed with 70% ethanol and then were dehydrated to 100% ethanol. Dried slides were counterstained with DAPI II (4',6-diamidino-2-phenylindole) (125 ng/mL). A total of 200 interphase nuclei were scored for each sample from donor sites 1, 3, and 5, as well as from host sites 2 and 6. Cells from site 4 were lost to contamination and were not assayed.
Chondrocyte Isolation and Expansion
The discarded tissues were rinsed with several changes of Hanks Balanced Salt Solution (HBSS) (Invitrogen, Carlsbad, California) containing 1% penicillin and streptomycin. Cartilage was dissected from three allograft sites and three corresponding adjacent host osteochondral sites (Figs. 1-A and 1-B). Cartilage from each site was removed from the subchondral bone with a sterile scalpel and minced into 1.5-mm fragments. Chondrocytes were released from the minced cartilage fragments by first rinsing them in 2.5% trypsin in Dulbecco Modified Eagle Medium (DMEM) for fifteen minutes at 37°C, followed by an overnight digestion in 2 mg/mL of type-IV bacterial collagenase (Sigma-Aldrich, St. Louis, Missouri) in DMEM with 2% calf serum and antibiotics in a shaking incubator set to 180 rpm and 37°C. The released cells were recovered by centrifugation at 1000 times gravity, rinsed once with DMEM, and counted with use of a hemocytometer. These cell populations were considered unpassaged (P0) and day 0 cells. For additional expansion, 1 × 105 cells were plated on tissue-culture-treated plastic and cultured for two weeks in DMEM containing 10% calf serum and antibiotics, with media changes every other day. To assess proliferation rates, cell numbers in subconfluent monolayer cultures were counted in a hemocytometer. Population doubling time (Td) was determined for the culture with use of Equation 1Equation 1 (below), where N0 is the number of seeded cells and N1 is the number of harvested cells.
Pellet Culture Chondrogenesis Assays
The chondrogenic capacity of isolated primary chondrocytes (P0) was compared with that of passaged (P2) cells. Three-dimensional cell pellet cultures were established in serum-free chondrogenic medium. Briefly, 2.5 × 105 cells were placed in 15-mL conical polypropylene centrifuge tubes and pelleted by centrifugation at 150 times gravity for five minutes. Medium was gently replaced with 500 μL of chondrogenic base medium (Lonza, Basel, Switzerland) containing 10 ng/mL of rhTGF-ß3 and an additional 300 ng/mL of rhBMP-7 (a gift from Dr. David Rueger, Stryker Biotech, Hopkinton, Massachusetts); the pellets were maintained in chondrogenic medium for up to fourteen days. The medium was changed every three to four days, and the growth factors were replenished with every medium change.
Gene Expression Analysis
RNA was isolated from 1 × 105 unpassaged (P0) host and allograft cells, and from seven-day pellet cultures with use of the RNeasy total RNA Kit (Qiagen, Valencia, California). cDNA was synthesized with use of the High Capacity Reverse Transcriptase Reagents, and real-time quantitative polymerase chain reaction (PCR) was performed with use of Assays-on-Demand TaqMan primers and probes and TaqMan reagents on an ABI Prism 7700 Sequence Detector (Applied BioSystems, Foster City, California). The TaqMan probe sets are shown in Table I. Assays were performed in triplicate and normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) levels with use of the recommended ΔCt method.
Histological and Immunohistochemical Staining
Three-dimensional pellets of P2 cells were harvested after fourteen days of culture in chondrogenic media. Pellets were fixed in Bouin fixative and rinsed with 70% ethanol overnight. Samples were embedded in paraffin, and 5-μm sections were cut. Sections were stained with hematoxylin and eosin and with 1% safranin-O to highlight cell morphology and sulfated glycosaminoglycans, respectively. Immunohistochemistry was performed with use of the antibodies shown in Table I. Commercially available antibodies were used at the recommended concentrations, and antibodies against cartilage oligomeric matrix protein (COMP) and a disintegrin-like and metalloprotease domain with thrombospondin motifs 7 (ADAMTS7) were generated in our laboratory and used as previously reported6,7. Detection was with peroxidase-conjugated secondary antibodies and 3,3’-diaminobenzidine (DAB) substrate according to the recommended protocols (ImmPRESS reagents and ImmPACT DAB; Vector Laboratories, Burlingame, California). Samples were counterstained with methyl green to identify cell nuclei for all sections except Sox9.
Source of Funding
This project was funded entirely by the Department of Orthopaedic Surgery, University of California Davis Medical Center.
Cell Identification
The allograft cells, which came from a male donor, were transplanted into a female patient at the time of surgery. In order to determine whether the cells recovered from the three allograft sites and the adjacent host sites were of male or female origin, isolated chondrocytes were assayed for the presence of sequences specific for the X and Y chromosomes. A total of 200 cells were scored from each site, and, without exception, only male cells were recovered from the allografts, while only female cells were recovered from the adjacent host tissue (Fig. 2).
Cell Proliferation
All of the isolated chondrocyte strains from host and allograft sources showed robust proliferation in culture. The proliferation rate during monolayer expansion differed between cells isolated from the host and those isolated from allografts, and varied from region to region (Fig. 3). Cells from the host tissue required an average (and standard deviation) of 11% ± 3% longer for population doubling than cells from the allograft tissue. There was also regional variation, with chondrocytes (from both host tissue and allograft) isolated from the trochlear groove proliferating the fastest. The average population doubling time of cells (host and allograft combined) from the condyles and the trochlear groove was 6.3 ± 0.6 days and 5.3 ± 0.5 days, respectively.
Gene Expression in P0 Chondrocytes
Gene expression was remarkably similar between cells from the host and allograft tissues at day 0 (Fig. 4). Extracellular matrix genes (aggrecan [Agc], Col2, and COMP) and the transcriptional regulator of chondrogenesis (Sox9) were all expressed at similar levels in chondrocytes isolated from host and allograft tissues. Matrix remodeling genes (ADAMTS7 and ADAMTS12) and a marker of hypertrophic differentiation (ColX) were also expressed at similar levels in chondrocytes isolated from host and allograft tissues. The exceptions were sites 1 and 2, in which there was somewhat greater variation between host and allograft in two of the eight genes tested—namely, the ColX level was higher in cells from allograft (site 1) than it was in cells from host (site 2) tissue, and ADAMTS12 showed the opposite trend. This is reflected in the larger error bars for these two genes (Fig. 4).
Chondrogenic Capacity
Chondrocyte strains from host and allograft tissues retained their chondrogenic capacity in an in vitro pellet culture assay, as measured by gene expression after seven days (Fig. 5). Gene expression profiles were very similar between pellets formed from host and those formed from allograft chondrocytes. There was high expression of matrix genes (Agc, Col2, and COMP) as well as the Sox9, and lower expression of ADAMTS7 and ADAMTS12, a gene expression profile consistent with chondrogenic differentiation of expanded cells. The exception was type-X collagen, which was more variable in pellets from allograft tissue than in pellets from host tissue sites. It was also noted that, despite the chondrogenic conditions used in the pellet cultures, the overall expression of matrix genes was lower than that in freshly isolated cells. Histochemical analysis of phenotype and matrix production after fourteen days in pellet culture revealed no apparent differences between cell pellets from host sources and those from allograft sources. The amount and localization of safranin-O-stained proteoglycans and the extracellular matrix components aggrecan and type-II collagen were similar between the host and allograft cell pellets, as were the amounts and locations of proteases ADAMTS12 and ADAMTS7 as well as the chondrogenic transcription factor, Sox9 (Fig. 6).
This study provides additional evidence of the clinical utility of fresh osteochondral allografts to treat cartilage defects. In our case, chondrocytes from the allograft tissue remained as an isolated population with a high viability, and they retained their gene expression profiles and proliferation capacities. The gene expression and chondrogenic potential of allograft and host cells were essentially identical after three years in vivo.
Disclosure: None of the authors received payments or services, either directly or indirectly (i.e., via his or her institution), from a third party in support of any aspect of this work. One or more of the authors, or his or her institution, has had a financial relationship, in the thirty-six months prior to submission of this work, with an entity in the biomedical arena that could be perceived to influence or have the potential to influence what is written in this work. No author has had any other relationships, or has engaged in any other activities, that could be perceived to influence or have the potential to influence what is written in this work. The complete Disclosures of Potential Conflicts of Interest submitted by authors are always provided with the online version of the article.