Keywords
cartilage, articular - allografts - bone transplantation - tissue and organ harvesting
- osteochondral lesion
Introduction
Osteochondral allograft transplantation (OAT), represents the only treatment option
capable of restoring bone stock and restoring joint surface with hyaline cartilage
after deep synovial joint damage.[1]
[2] The main indication for OAT is the presence of focal osteochondral lesion > 2.0 cm2 associated with painful symptoms in the load-bearing joints.[1]
[2]
[3]
[4] Preservation of human osteochondral tissue is a key factor in OAT success. Tissue
storage time, as well as the preservation means used, are related to chondrocyte viability
maintenance after the procedure.[1]
[5]
The 4°C temperature is the most commonly used for preserving osteochondral tissue
in humans or animal models.[6] Tissue cooling below the average body temperature of 37°C induces a reduction in
chondrocyte cellular metabolism, leading to lower consumption of nutrients provided
by the preservation medium used.[7] To date, there is no consensus in the literature regarding the standardization of
a protocol for the harvest, transport and preservation of osteochondral tissue in
tissue banks (TBs), either national or in those located in other countries in the
Americas and Europe. The aim of the present study was to develop a protocol for the
harvesting, transport and preservation of human osteochondral tissue for use in TBs.
Material and Methods
Selection of Donors
The study population consisted of 5 musculoskeletal tissues from corpse donors (CDs)
from June 2015 to December 2016 that met the criteria for organ donation of the National
Transplant System (SNT, in the Portuguese acronym), according to the rules published
by the Ministry of Health (MS, in the Portuguese acronym) Ordinance n° 2.600 published
on October 21, 2009. After approval by the Institutional Research Ethics Committee
(CAAE 45288015.7.0000.5273), CDs aged between 15 and 45 years old, without history
of trauma, surgery or previous knee joint infection, were included in the study, selected
for harvesting and removal of osteochondral fragments. The collected joint was removed
in its entirety, and taken to the TB, where it was processed and submitted to visual
evaluation, being excluded in case of any articular cartilage injury. Cadaveric donors
from outside the state of Rio de Janeiro were also excluded due to the long-lasting
travel time between the harvesting site and the headquarters of the TB. Finally, the
knees of five CDs were selected for the research ([Figure 1]).
Fig. 1 Corpse donor selection flowchart for research. CD (corpse donor).
Harvesting and Transport Protocol
After median knee incision and subcutaneous dissection, femoral osteotomies were performed
2.0 cm above the superior pole of the patella, and tibial, 5 cm distal to the tibial
tubercle, with the aid of a surgical oscillating saw, without opening the knee joint
capsule. The joint was immersed in Ringer Lactate, vacuum packaged, and stored in
a thermal flask at 4°C, controlled by a local thermostat until arrival at the TB ([Figure 2]).
Fig. 2 Thermal flask for transporting osteochondral tissue (A). Temperature control thermometer
(B). Source: Personal Archive.
Human Osteochondral Tissue Processing Protocol
Tissue processing was performed by a medical staff member of the research team in
an ISO 5-class room located within the TB. Eight 2 × 3 cm2 fragments were collected by CD, for histological analysis, which were preserved at
4°C. The preservation medium used was composed by cell culture medium without Iscove's
serum (Thermo Fischer, Waltham, MA, USA) supplemented with 10% human albumin and vancomycin
100 µg/mL. Tissues were subjected to analysis on the harvesting day, defined as day
zero (d0), and after 15 (d15), 30 (d30) and 45 days (d45) of preservation ([Figure 3]). Tissue fragments were also sent for culture in the microbiology laboratory of
the institution to exclude infection.
Fig. 3 Methodology of preparation of osteochondral fragments (OF) for tissue analysis. Distal
femoral joint surface selected for OF removal and histological analysis (A). The fragments
were kept in the preservation medium at 4°C. (B). Sample Evaluation Period (C). D
(day). Source: Personal Archive.
Tissue Analysis Protocol
The osteochondral fragments were submitted to histological sections and stained by
hematoxylin and eosin (H&E) technique for global tissue analysis, and by Safranin
O (SO) for glycosaminoglycan deposition evaluation. The slides were analyzed under
an optical microscope and photographed to record the results at 100x magnification.
The generated images were named alphanumerically, so that the letter indicated the
preservation time of the tissue, and the number indicated the donor, so that the evaluators
were blinded to the preservation time and the CD. The evaluators received the images
obtained in an electronic storage device, and the application forms of the two selected
scores for the research. We used the modified Mankin score,[8] whose score ranges from 0 to 14 points, and the score described by the OsteoArthritis
Research Society International (OARSI),[9] which quantifies the changes related to synovial cartilage degeneration, graduating
them from 0 to 6.
Analysis of the Results
The results obtained were tabulated in a Microsoft Excel (Microsoft Corporation, Redmond,
WA, USA) spreadsheet for further statistical analysis. The analysis of the present
study was formed by the scores obtained from the visual evaluation of the osteochondral
fragments in the 4 evaluation moments (d0, d15, d30, d45) according to the 2 previously
selected scores. In all, 38 evaluations were performed, which make up the database
dimension of the present study. One assessment was lost regarding the CD 388 in time
d0. The collected data were analyzed by the IBM SPSS Statistics for Windows, Version
22.0 (IBM Corp. Armonk, NY, USA). The graphics were built in the Microsoft Excel 2011
software.
For sample characterization and descriptive analysis of the variables, given the qualitative
nature of the scores, the mean and mean-based statistics could not be calculated;
thus, the data were synthesized by means of relative frequency distributions, and
descriptive statistics as median, minimum and maximum. Following the nonparametric
approach of analysis, given the ordinal qualitative nature of the scores, the significance
of the temporal evolution of a score was assessed by the Wilcoxon test, comparing
the scores of one assessment with the respective scores of the previous assessment.
All of the comparisons were performed considering a maximum significance level of
5% (0.05), that is, the following decision rule was adopted in the tests: rejection
of the null hypothesis whenever the p-value associated with the test was < 0.05.
Results
Histological Evaluation
Sections stained by H&E of the tissue from the harvesting day (d0), and from the 15th day of preservation (d15), showed no structural changes in the superficial layer
of the articular cartilage ([Figure 4A-B]). However, in samples evaluated after 30 days (d30), and after 45 days (d45) of
preservation, changes were identified in the superficial layer of the cartilage, such
as loss of continuity and presence of cracks in the articular surface. ([Figure 4C-D]).
Fig. 4 Photomicrograph of the preserved superficial cartilage layer at 4°C stained with
hematoxylin and eosin (H&E). H&E-stained histological sections of osteochondral fragment
obtained on the day of tissue harvesting (A), histological section of osteochondral
fragment after 15 days of preservation, d15 (B), histological section of osteochondral
fragment after 30 days of preservation, d30 (C) and histological section of osteochondral
fragment after 45 days of preservation, d45 (D).
When histologically evaluating the intermediate cartilage region by H&E staining,
no structural changes were observed in the four evaluation moments. The deep cartilage
layer as well as the subchondral bone plate remained preserved throughout the preservation
period analyzed.
Analysis of histological sections stained with SO after 15 days of preservation (d15)
showed a decrease in the concentration of proteoglycans in the peripheral portion
of the superficial layer when compared with the samples in d0. After 30 days of preservation
(d30), there was a decrease in proteoglycan concentration in the superficial cartilage
layer of the analyzed osteochondral fragment (OF). Finally, after 45 days of preservation
(d45), structural diffuse lesions in the superficial layer of cartilage were observed,
associated with decrease in concentration of proteoglycans ([Figure 5]). Histological analysis after SO staining did not identify a decrease in proteoglycan
concentration in the intermediate cartilage layer, nor in the deep layer, in the four
evaluation times.
Fig. 5 Photomicrograph of the superficial cartilage layer preserved at 4°C and stained with
Safranin O (SO). Histological section stained with SO from osteochondral fragment
obtained on the day of tissue harvest, d0 (A), histological section of osteochondral
fragment after 15 days of preservation, d15 (B), histological section of osteochondral
fragment after 30 days of preservation, d30 (C) and histological section of osteochondral
fragment after 45 days of preservation, d45 (D).
Result of Histological Scores
The frequency distribution of the results obtained according to the Mankin score is
presented in [Figure 6]. The highest score obtained was five points, regardless of the period analyzed.
After 15 days of preservation, 100% of the evaluated slides obtained 3 points out
of the 14 possible points, showing good results regarding the tissue alterations analyzed
by the score. After 45 days of preservation, the results were similar, with 90% of
the sections evaluated adding up to 3 points.
Fig. 6 Frequencies of results according to the Mankin histological score. The maximum score
obtained on the Mankin score was 5 points.
At 30 days of preservation, the samples had a statistically significant difference
in relation to the 15-days-preserved tissue, suggesting worse quality of osteochondral
tissue according to the criteria used by the Mankin score.
The frequency distribution obtained according to the OARSI score is presented in [Figure 7]. In the period defined as d0, 30% of the evaluated slides presented histological
alterations even before the samples were submitted to preservation at 4°C. The alterations
described were restricted to the superficial layer of the articular cartilage, especially
the decrease of proteoglycan concentration. At 15 days of preservation, 80% of the
samples were classified as type 0 or 1, containing only superficial changes in the
tissue according to the description of the score used. After 30 days of preservation,
70% of the samples were classified as type 2, containing structural changes in the
superficial layer, as well as a decrease in proteoglycan concentration. No tissue
sample analyzed showed changes related to articular cartilage degeneration, classified
as type 4, 5 or 6 according to the OARSI score.
Fig. 7 Osteoarthritis Research Society International. Abbreviations: OARSI, Osteoarthritis
Research Society International.
According to the histological evaluation assessed by the OARSI score, no statistically
significant variations were observed between the results obtained in the different
periods of osteochondral tissue evaluation.
Discussion
Osteochondral allograft transplantation represents a biological method capable of
providing hyaline cartilage for the treatment of osteochondral lesions thicker than
2cm2.[1]
[2]
[4]
[10] Cell viability of donor tissue at the time of surgery is an indispensable factor
for successful treatment. The absence of a protocol defined in the literature led
us to the need to develop a proper methodology for harvest, transport and storage
of human osteochondral tissue.
Several factors influence the preservation of human osteochondral fragments, aiming
at increasing the number of viable chondrocytes as well as the longer period of tissue
storage. Thus, temperature and storage medium are variables widely discussed in the
literature.
Regarding temperature, the discussion involves preservation at 4°C, 25°C, 37°C or
cryopreservation.[11]
[12]
[13]
[14]
[15]
[16]
[17] The increase in preservation temperature is associated with a higher nutrient consumption
offered by the preservation medium, there is the need for periodic change of medium,
exposing the tissue to the risk of contamination and increasing the cost of storage.[12] Cook et al[17] were able to prove up to 89.8% of viable chondrocytes in canine osteochondral fragments
using the Missouri Osteochondral Allograft Preservation System (MOPS) at 25°C after
60 days of tissue preservation; however, fresh preservation at 4°C remains the gold
standard in the literature.
In a preliminary study, performed with surgical disposal material, our group showed
that refrigeration at 4°C offers better preservation capacity of osteochondral fragments
than at 37°C for up to 14 days.[18] For this reason, in the current study with young CD tissues, we kept the option
of cooling the fragments to 4°C. Our results confirm previous findings that the temperature
of 4°C is adequate for allograft preservation.
Several supplemented media were evaluated for their benefits on the viability of preserved
cartilaginous tissue, including dexamethasone-associated media,[19] hyaluronic acid,[20] allogeneic serum,[21] or tumor necrosis factor inhibitor α[19] and the cellular apoptosis inhibiting agent,[22] among others. The divergence in the literature between the means used, the absence
of in vivo research, and the high cost of the preservation methods evaluated, make
it difficult to decide on the best supplement to use in conjunction with preservation
media to improve the quality of tissue stored for transplantation. Pearsall et al[7] determined that human osteochondral allografts could be kept viable for up to 44
days with an average viability of 67% when kept in a storage medium supplemented with
antibiotic, glutamine and 10% fetal bovine serum, without growth factors. Another
study concluded that allografts stored in antibiotic Ringer lactate solution could
be implanted within 7 days, while storage in serum-free medium would increase this
time to 2 weeks.[17] Considering the findings of these studies, we opted to include in our protocol transporting
the fragments in Ringer lactate, because it is cheaper and affordable in any hospital
where the harvest of tissue is made, leaving the preservation in supplemented-Iscove's
medium, more expensive and dependent of structure for preparation, for storage after
processing by the TB.
The method described for the transport of osteochondral tissue kept it at the appropriate
temperature (4/10°C) until the beginning of the tissue processing procedure. Preservation
of osteochondral tissue at 4°C was able to maintain the structural characteristics
of the articular cartilage layers for up to 15 days of storage in cell culture medium
without supplementation with Iscove's serum, supplemented with 10% human albumin and
vancomycin 100 µg/mL.
Histological evaluation using H&E-stained slides demonstrated preservation of articular
cartilage layers removed on the day of tissue collection (d0), being these defined
as control group. After 30 and 45 days of preservation, there were changes in the
superficial layer of the tissue, demonstrating that this region is subject to changes
related to the preservation time. The integrity of the superficial portion of the
cartilaginous tissue is of utmost importance for load absorption and distribution.
Lesions in the superficial cartilage layer are related to alteration in tissue permeability,
favoring the penetration of synovial fluid, initiating the process of joint degeneration.[23] The layers of intermediate, deep cartilage and calcified cartilage did not change
during the period evaluated. These findings describe a typical pattern of injury related
to the preservation of human osteochondral tissue, in which only the superficial layer
of cartilage presented discontinuities and delamination.
Visual evaluation of SO-stained slides showed a gradual decrease in proteoglycan concentration
in the extracellular matrix along with the increase of preservation time in the studied
samples. In 30% of the samples there was a decrease in the concentration of proteoglycans
in the superficial layer of the tissue soon after harvesting (d0). This finding suggests
that alterations in chondrocyte metabolism may exist even in physiologically young
patients without lesions described in the articular cartilage. After 30 days of preservation,
all of the analyzed samples showed a decrease in proteoglycan concentration in the
superficial layer. The remaining articular cartilage layers had their proteoglycan
concentration preserved after the entire evaluation period (45 days). Our results
confirm the need for osteochondral tissue transplantation with the shortest possible
preservation time, because they describe the structural alteration after fresh OF
preservation, although restricted to the superficial layer of the articular cartilage.
Structural changes in the superficial layer allow the flow of synovial fluid, leading
to the formation of subchondral cysts, thus initiating the process of joint degeneration.[21]
Our work had some limitations. First, there were no quantitative analyses of cell
viability of the tissue studied. Second, the histological evaluation methodology used
was based on scores created for the study of cartilaginous tissue with degenerative
changes. There is no histological score in the literature to evaluate cadaveric human
osteochondral tissue. Finally, the data generated are of a qualitative nature and
may be subject to interobserver variations.
Conclusion
The described protocol defined the transport of a knee in a block immersed in Ringer
Lactate at 10°C controlled temperature until its arrival at the TB. After processing,
the preservative solution was composed of cell culture medium without Iscove's serum,
supplemented with 10% human albumin and vancomycin 100 µg/mL. Tissue was preserved
at 4°C until transplantation, characterizing fresh preservation.
