Atwenty-two-year-old man with a large defect on the articular surface of
the lateral femoral condyle of the knee underwent fresh osteochondral
allografting from a female donor. He recovered well from the allograft
procedure and had a relatively pain-free knee for the next twenty-five years.
He presented to us with knee pain on the lateral side and tricompartmental
arthritis predominantly in the lateral compartment
(Fig. 1). A magnetic resonance
imaging scan was acquired, and it demonstrated a tear of the posterior horn of
the lateral meniscus with loss of the articular cartilage thickness along the
posterior aspect of the lateral femoral condyle
(Fig. 2). He ultimately
requested total knee arthroplasty because of the unremitting knee pain. The
procedure was performed exactly twenty-nine ye ars after the implantation of
the fresh osteochondral allograft. At the time of the procedure, he had a
sclerotic region on the distal aspect of the lateral femoral condyle with a
small amount of remaining cartilage. No demarcation could be seen between the
host bone and the allograft. Scrapings were obtained from the remaining
articular cartilage along the posterior aspect of the lateral femoral condyle,
and the bone resected from this region underwent histological analysis. Our
hypothesis was that chondrocytes from the allograft donor remained alive and
active in the patient over the twenty-nine-year interval since the
procedure.
Analysis of Osteochondral Tissue and Remaining Articular
Cartilage
Scrapings of the remaining cartilage from the posterior aspect of the
lateral femoral condyle were obtained at the time of total knee arthroplasty.
Fluorescence in situ hybridization (FISH) and karyotype analysis were
performed on cultured chondrocytes from these scrapings. Osteochondral tissue
from this region was resected during the total knee replacement. This tissue
was fixed with 10% formalin, decalcified with 10% formic acid, and then held
in 70% EtOH. The sections were embedded in paraffin and stained with
hematoxylin and eosin.
For cytogenetic analysis, femoral cartilage tissue was minced by scalpel
dissection and dissociated with collagenase (Type IV; Sigma, St. Louis,
Missouri). The cells were cultured for ten days in tumor media consisting of
20% fetal bovine serum (Gemini Bio-Products, West Sacramento, California),
RPMI 1640 (Invitrogen, Carlsbad, California), and 100X antibiotic-antimycotic
(Invitrogen). Cells were treated with colcemid (Invitrogen) and hypotonic KCl
solution and were fixed in methanol acetic acid. Chromosomes were banded with
use of the standard trypsin-Giemsa method. Karyotypes were described according
to the International System for Human Cytogenetic Nomenclature (ISCN
1995)2.
Fixed cells from cultured cartilage tissue were dropped onto glass slides
and dried. Dual-color fluorescence in situ hybridization was performed on the
cells with the Vysis Chromosome Enumeration Probe (CEP) X SpectrumGreen and
CEP Y SpectrumOrange probes (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. Male cells
(XY) demonstrate one green and one orange signal. The cells were denatured and
hybridized overnight with use of a HYBrite system (Vysis). The slides were
washed with 70% EtOH and then were dehydrated to 100% EtOH. Dried slides were
counter-stained with DAPI II (4',6-diamidino-2-phenylindole) (125
ng/mL). A total of 206 interphase nuclei were scored.
DNA was extracted with use of Puregene reagents (Gentra Systems;
Minneapolis, Minnesota) and was amplified with primers specific for the
amelogenin gene on the X chromo-some and the amelogenin-like sequence on the Y
chromo-some3,4.
Polymerase chain reaction products were visualized in ethidium bromide-stained
agarose gels after electrophoresis (data not shown).
The results of histological analysis by hematoxylin and eosin staining of
the posterior aspect of the lateral femoral condyle demonstrated severe
thinning of the articular cartilage with areas of complete cartilage loss and
exposed subchondral bone (Fig.
3). These findings were consistent with severe osteoarthritis in
this anatomical location. Gender identification by polymerase chain reaction
established that the DNA extracted from the lateral femoral condyle had both X
and Y-specific fragments. Since both X and Y-specific fragments were present
and the polymerase chain reaction was not quantitative, it could not be
determined whether the DNA was exclusively male or a mixture of male and
female.
Fluorescence in situ hybridization demonstrated that 138 (67%) of the 206
interphase nuclei were XX and sixty-eight (33%) were XY
(Fig. 4). Routine chromosome
analysis showed nine of sixteen metaphase cells were 46,XX, and seven of
sixteen metaphase cells were 46,XY.
This report is the first, as far as we know, to confirm donor cell survival
in a fresh human osteochondral allograft. More importantly, the female
chondrocytes or their progeny remained intact for nearly thirty years in vivo
with no systemic immunosuppression.
A number of strategies have been used to study the in vivo cell viability
of fresh osteochondral grafts. Czitrom et al. used autoradiography to study
the articular cartilage of allografts biopsied after
transplantation5.
They found chondrocyte viability from 37% to 99% at intervals ranging from one
to six years. Although chondrocytes are considered relatively immobile cells
within the matrix, the origin of these cells as donor cells was not
definitively ascertained.
Convery et al. removed a fresh osteochondral allograft from a medial
femoral condyle eight years after
implantation6. They
studied decalcified sections of the specimen by standard histological analysis
as well as by vital staining and demonstrated a mixture of live and dead
cells. Once again, the origin of the cells could not be definitively
determined. McGoveran et al. analyzed a biopsy specimen obtained seventeen
years after an osteoarticular distal femoral allograft
replacement7.
Standard light microscopy revealed normal cartilage thickness, a number of
focally empty cartilage lacunae, and areas of chondrocyte clumping presumably
due to chondrocyte regeneration. Examination with electron microscopy
confirmed intact rough endoplasmic reticulum, mitochondria, and cellular
membranes. Again, however, the cellular origin of these chondrocytes was not
definitively determined.
Strategies similar to our methodology have been used to determine the
survival of limbic stem cells transplanted for corneal dysfunction.
Fluorescent in situ hybridization analysis was used by Shimazaki et al. to
demonstrate survival of donor cells after limbic stem cell transplantation at
2.4 years after the
procedure8. However,
the patient in their study was treated with dexamethasone for three weeks and
cyclosporine for six months, an important distinction from the patient in the
present study in whom no immunosuppression was used.
The findings of the current report highlight the presence of a mixed
population of cells after fresh osteochondral allografting. Furthermore, this
report confirms the long-term survival of donor cells from a nonvascularized
osteochondral allograft with no systemic immunosuppression at nearly three
decades after transplantation. ?