High‐Speed Centrifugation Efficiently Removes Immunogenic Elements in Osteochondral Allografts

Objectives The current clinical pulse lavage technique for flushing fresh osteochondral allografts (OCAs) to remove immunogenic elements from the subchondral bone is ineffective. This study aimed to identify the optimal method for removing immunogenic elements from OCAs. Methods We examined five methods for the physical removal of immunogenic elements from OCAs from the femoral condyle of porcine knees. We distributed the OCAs randomly into the following seven groups: (1) control, (2) saline, (3) ultrasound, (4) vortex vibration (VV), (5) low‐pulse lavage (LPL), (6) high‐pulse lavage (HPL), and (7) high‐speed centrifugation (HSC). OCAs were evaluated using weight measurement, micro‐computed tomography (micro‐CT), macroscopic and histological evaluation, DNA quantification, and chondrocyte activity testing. Additionally, the subchondral bone was zoned to assess the bone marrow and nucleated cell contents. One‐way ANOVA and paired two‐tailed Student's t‐test are used for statistical analysis. Results Histological evaluation and DNA quantification showed no significant reduction in marrow elements compared to the control group after the OCAs were treated with saline, ultrasound, or VV treatments; however, there was a significant reduction in marrow elements after LPL, HPL, and HSC treatments. Furthermore, HSC more effectively reduced the marrow elements of OCAs in the middle and deep zones compared with LPL (p < 0.0001) and HPL (p < 0.0001). Macroscopic evaluation revealed a significant reduction in blood, lipid, and marrow elements in the subchondral bone after HSC. Micro‐CT, histological analyses, and chondrocyte viability results showed that HSC did not damage the subchondral bone and cartilage; however, LPL and HPL may damage the subchondral bone. Conclusion HSC may play an important role in decreasing immunogenicity and therefore potentially increasing the success of OCA transplantation.


Introduction
A rticular cartilage (AC) defects are a common disorder affecting people of all ages; however, their treatment remains challenging. 1As cartilage lacks nerves, lymphatic vessels, and blood vessels, it is unable to heal itself. 2,3In the aging population, cartilage or osteochondral lesions can be successfully treated by arthroplasty; however, arthroplasty is not acceptable for young and active patients. 4,56][7][8] The success of fresh OCA transplantation relies on the provision of viable cartilage and stable subchondral bone; however, failure can still occur.][16][17] The main factors contributing to the immunogenicity of grafts are allograft cartilage and subchondral bone. 18Chondrocytes have immunogenic elements; however, the special composition and structure of the extracellular matrix protect them from the recognition of chondrocyte surface antigens by the host immune system, thereby allowing them to successfully avoid rejection. 4,15Additionally, the subchondral bone contains many bone marrow cells, blood cells, lipids, and proteins, which are mostly immunogenic. 18,191][22][23][24] However, the effectiveness of PL has been questioned; it did not significantly reduce the bone marrow content of OCAs, possibly because it could not remove the deep bone marrow elements. 19Thus, the removal of immunogenic elements of OCAs requires better methods during pre-transplantation.
In this study, we hypothesized that five such methods could physically remove the immunogenic elements of OCAs.The vortex vibration (VV) method removes blood and lipids from the graft by inducing an oscillatory effect through high-speed rotation; the ultrasound method uses high-frequency vibrations to remove immunogenic elements from bone tissue and pore surfaces; 25 and the centrifugation method uses the centrifugal force of high-speed rotation to separate the blood, bone marrow, and lipids from the inside of the graft. 26,27ence, the purpose of the study was to (i) validate whether the various methods were feasible, whether the chondrocyte viability and extracellular matrix were affected, and whether trabecular bone structure was destroyed after treatment of OCAs and to (ii) evaluate the effectiveness of each method for removing immunogenic elements from OCAs to identify an optimal method.

Weight Assessment
For each control and experimental group, five OCAs were assessed for dry weight as an indicator of the weight of bone marrow elements after removal.The OCAs were collected and immediately frozen at À80 C for 4 hours, placed in a vacuum freeze drier (Alpha 1-4, Christ, Germany), and then processed according to standard drying procedures.After 24 h, the OCAs were measured using an electronic balance.

Micro-Computed Tomography (micro-CT) Evaluation
For the six experimental groups, both pre-and posttreatment samples were scanned by a micro-CT system (vivaCT80, SCANCO Medical AG, Switzerland) at high resolution with four OCAs per group. 31OCAs were scanned, and successive micro-CT two-dimensional (2D) images were obtained with an image resolution of 1024 Â 1024 with a pixel size of 20 Â 20 μm, and a layer spacing of 15.6 μm.The OCA was rinsed with sterile saline for 2 min and rotated every 10 s.Per plug, 1 L saline was used Ultrasound (n = 21) The OCA was placed in 50-mL centrifuge tubes with 30 mL of sterile saline and underwent highfrequency ultrasound treatment (Scientz, Ningbo, China): frequency of 40 kHz and 2 min time.VV (n = 21) The OCA was placed in 50-mL centrifuge tubes with 30 mL of sterile saline and shaken for 2 min (3000 r/min) using a vortex mixer (Thermo Scientific, Massachusetts, USA).LPL (n = 21) The OCA was irrigated using conventional pulse lavage equipment (Clean, Guangzhou, China).The sample was rinsed with saline as close to the spray tip as possible, from the base of the subchondral bone upwards, rotating every 10 s, for 2 min, at a flow rate of 1100 mL/min.HPL (n = 21) Same as the method described above, with a lavage flow rate of 1260 mL/min.HSC (n = 21) The OCA was placed into 1.5-mL centrifuge tubes and centrifuged for 2 min at 12000 r/min (5430R, Eppendorf, Germany).
The subchondral bone was defined as a region of interest, and the images were analyzed using Scanco software.The bone volume per tissue volume (BV/TV, %), connection density of the bone trabeculae (Conn.D, 1/mm 3 ), trabecular number (Tb.N, 1/mm), trabecular space (Tb.Sp, mm), and bone mineral density (BMD) (mg HA/ccm) were analyzed. 32

Macroscopic Evaluation
For each control and experimental group, the gross appearance of four OCAs was observed to evaluate cleansing efficacy.The OCAs were fixed in 4% paraformaldehyde for 48 h and decalcified using 10% EDTA; after 1 week, the OCAs were evenly divided into two groups using a scalpel and evaluated for gross appearance.

Histological Analyses
For each control and experimental group, four OCAs were histologically analyzed to evaluate the effectiveness of the removed bone marrow elements and the completeness of the cartilage.As described above, the OCAs were evaluated macroscopically and subjected to EDTA decalcification.One month later, the OCAs were dehydrated, embedded in paraffin, and sectioned at 5-mm intervals according to recognized histopathological standards.Sections were stained with hematoxylin and eosin (HE) (Solarbio, Peking, China) to analyze the effectiveness of the removed bone marrow elements in the subchondral bone, with safranin O and fast green (Saf O) (Solarbio, Peking, China) used to assess glycosaminoglycan (GAG) in the cartilage, and Sirius red (Solarbio, Peking, China) used to detect collagen (COL) in the cartilage.Each sample was divided into the following three zones for analysis: superficial (external third), middle (middle third), and deep (central third) (Figure 1A). 19The effectiveness of removed bone marrow elements was expressed as a "blank cavity area ratio" technique (Figure 1B).The blank cavity area ratio, which is the area of the blank bone marrow cavity within the bone trabeculae as a percentage of the total area of the bone trabeculae and bone marrow cavity, was calculated using ImageJ software (version 1.51 k). 23

DNA Quantification
For each control and experimental group, four subchondral bones were examined for DNA concentration to determine the number of remaining nucleated cells. 19The tissue was obtained from the superficial, middle, and deep zones of the subchondral bone, each sample weighing 30 mg.After cutting and grinding, all tissues were incubated in 20 μL lysis buffer and shaken gently at 56 C for 3 hours.Subsequently, DNA was extracted according to the manufacturer's instructions of the TIANamp Genomic DNA Kit (Tiangen, Peking, China) as follows: 200 μL of buffer solution was added, mixed thoroughly, and incubated at 70 C for 10 min to allow the solution to become clear.Later, 200 μL of anhydrous ethanol was added and mixed again.The resulting solution and precipitate were moved into the adsorption column, and the specific solution was repeatedly added to rinse.Finally, 100 μL buffer was added to obtain DNA.The DNA concentration was measured using a NanoDrop One spectrophotometer (Thermo Scientific, Massachusetts, USA) at a wavelength of 260 nm.Nucleic acid purity was assessed using the absorbance ratio A260/A280 nm. 19

Chondrocyte Viability
For each control and experimental group, four OCAs were stained for live and dead cells to assess chondrocyte viability. 33,34The subchondral bone was removed from the graft using a scalpel.The cartilage was adhered to the specimen table and sectioned in cross-section using a vibratome (VT 1200S, Leica, Germany) to obtain 50-um thickness cartilage slices.Each cartilage slice was stained for two-color fluorescence; the live cell stain calcein-AM (green color, Bestbio, China) and the dead stain propidium iodide (PI, red color, A B Bestbio, China) were used for 40 minutes in a water bath at 37 C. Cartilage slices in the deep zone were imaged using a confocal laser-scanning microscope (Leica, Germany).The ImageJ software was then used to count the cells in the images, and chondrocyte viability was calculated as the percentage of live cells relative to the total number of cells.In each cartilage slice, the chondrocyte viability was determined as the percentage of green cells relative to the total number of green and red cells. 28,35The experimental procedures were repeated two times.

Statistical Analyses
The data are presented as the mean AE standard deviation and statistically analyzed using SPSS 25.0 (IBM, NY, USA) and GraphPad Prism 9 (Prism, CA, USA).Weight assessment, histological analyses, DNA quantification, and chondrocyte viability of OCAs were compared between the control and experimental groups using a one-way ANOVA of variance with a Bonferroni's post hoc test.Micro-CT evaluations of both pre-and post-treatment of OCAs were compared using a paired two-tailed Student's T-test.Statistical significance was set at p < 0.05.

Weight Assessment
In the dry weight assessment, there were no significant differences in the saline, ultrasound, and VV-treated OCA groups compared to the control group; however, significant differences were observed in the LPL (p = 0.0009), HPL (p < 0.0001), and HSC (p = 0.0021) groups.The HPL (0.25 AE 0.12 g) group had the lowest dry weight of OCAs, followed by the LPL (0.28 AE 0.04 g) and HSC (0.29 AE 0.01 g) groups (Figure 2).

Micro-CT Findings
The micro-CT results from the six experimental groups were analyzed (Table 2).We observed no significant differences in the pre-and post-treatment CT results in the saline, ultrasound, VV, or HSC groups; however, significant differences were observed in the pre-and post-treatment CT results in the LPL and HPL groups.Additionally, there were significant differences in the pre-and post-treatment results between BV/TV ( p = 0.0494), Tb.N ( p = 0.0060), and Tb.Sp (p = 0.0145) in the LPL group, and there were significant differences between Conn.D (p = 0.0355), Tb.N (p = 0.0017), Tb.Sp (p < 0.0001), and BMD (p = 0.0375) in the pre-and post-treatment results of the HPL group (Table 2).Furthermore, micro-CT images showed sparse bone trabeculae in the superficial zone of OCAs after LPL and HPL treatments compared to the pre-treatment OCAs (Figure 3).

Macroscopic Observations
Gross photographs of the OCAs showed that the trabeculae in the control group were all black, whereas in the saline, ultrasound, and VV groups, the peripheral trabeculae were yellow, and the central trabeculae were black.However, in the LPL and HPL groups, the peripheral trabeculae were white, and the central trabeculae were black.The most common characteristic was white trabeculae in the HSC group.Therefore, the HSC group exhibited the best macroscopic cleaning effect (Figure 4).

DNA Quantification
The DNA concentration was also analyzed in the different zones of the subchondral bone (Figure 6).(p = 0.0004), and 65.15% ( p < 0.0001).Additionally, the DNA concentration in the HSC group was lower than that in the LPL (p = 0.0044) and HPL ( p = 0.0053) groups.However, there were no significant differences in DNA concentrations among the saline, ultrasound, VV, or control groups (Figure 6).

Chondrocyte Viability
The staining results of live and dead cells showed that chondrocyte viability was higher than 80% in each group, although there was no statistical difference (Figure 7).

Discussion
The results of this study indicated that HSC is the optimal pre-transplant OCA treatment method that can reduce most immunogenic elements without destroying bone trabeculae and impairing chondrocyte activity and the extracellular matrix.Furthermore, our data showed the following: first, chondrocyte viability and extracellular matrix were not negatively affected after five methods of treating OCAs, but the PL technique may destroy bone trabecular structures; second, both PL and HSC can reduce the antigenic elements in the subchondral bone, with HSC being more effective in removing the superficial, medium, and deep zones antigenic elements of the subchondral bone.Additionally, after treatment of OCAs with HSC method, the bone-marrow cavity blanking area could reach more than 60%, DAN content was reduced by 65%, and most of the bone marrow, lipids, and blood were removed.
Importance of the Immunogenic Elements Removal in OCA OCA transplantation presents an extremely special case among allogeneic transplant procedures because it does not require ABO blood type or human leukocyte antigen matching before transplantation. 19However, some OCA treatments before transplantation are necessary to reduce the entry of heterologous genes into the host.Moreover, as it is necessary to preserve chondrocyte activity and the integrity of the extracellular matrix of OCAs, chemical treatment is not an option, and physical treatment is preferred.Generally, the success of OCA transplantation depends primarily on the integration of the graft-host defect interface zone, where the integration and remodeling of the donor and host subchondral bone meet the biomechanical and biological requirements.Therefore, promoting the integration of the graft and host subchondral bone in the early posttransplantation period is crucial.7][38] The immune rejection reaction has been confirmed in the primary subchondral bone after clinical OCA failure. 12,13,18Van Dijk proposed to minimize the bony portion of the graft, since creeping substitution is less effective over larger distances in thicker grafts. 39Additionally, overlong or excessive subchondral bone carries more immunogenic elements to produce a stronger immune response and does not allow for effective osseous integration. 17,18,20,40 related study discovered that OCAs >9 mm thick were prone to bone cracks, which may be unfavorable for osseous integration.6 However, Babu et al. 20 demonstrated that OCAs of 7 mm thickness resisted pullout significantly more than those of 4 mm thickness.Ackermann et al. 36 discovered that OCAs with thinner thicknesses were more prone to subchondral cyst formation at the graft-host junction, with bone thickness <5 mm increasing the risk of cystic degeneration nearly five-fold.In this study, a subchondral bone thickness of 6 mm was used, consistent with the ideal subchondral bone thickness (3-6 mm).6,36 Limitations of PL Technique in OCA PL technique is often used clinically to thoroughly wash the subchondral bone to reduce the blood and bone marrow content of the donor source.4,36,41 Meyer et al. 23 proposed that combination saline and high-pressure carbon dioxide lavage is more effective in removing bone marrow elements than saline PL alone. Sn et al. 24 demonstrated that the effect of PL on fresh OCAs depends on irrigation intensity and time. Hoever, Ambra et al. 19 believed that PL could not effectively reduce the bone marrow content in OCAs; it could only wash the bone marrow in the superficial zone of the graft and might even concentrate the superficial bone marrow in the deep zone as the DNA content of OCAs did not show a significant decrease after PL.In this study, DNA quantification revealed that PL significantly reduced the bone marrow concentration in the superficial zone.Additionally, the gross photographs showed that the central area of the LPL and HPL groups was black and darker than that found in the saline, ultrasound, and VV groups (Figure 4).The authors attributed this to the fact that PL concentrates blood and bone marrow from the superficial zone of the OCAs in the deeper zone.Weight assessment showed a significant decrease in the weight of OCAs in the LPL and HPL groups; however, micro-CT results revealed a significant difference between pre-and post-treatment with PL treatment (Table 2).Additionally, micro-CT images showed sparse bone trabeculae in the superficial zone of OCAs after LPL and HPL treatments compared to the pre-treatment OCAs (Figure 3). Weattributed this to the fact that PL disrupts the superficial trabeculae of the subchondral bone, resulting in significant weight loss.
Effectiveness and Potential of HSC Method in OCA Centrifugation was often used to collect bone marrow from long bones. 26,27This was the first study to use HSC to remove immunogenic elements from the subchondral bone.Gross photographs showed that the subchondral bone of the OCAs became white after 2 min, compared with the black color of the subchondral bone in the control group, and the black or yellow color of the subchondral bone in the other experimental group (Figure 4).Therefore, the authors concluded that HSC completely removed the blood element and effectively removed the lipid element.The lipid was present and appeared yellow in the bone trabeculae and in the degreased bones it became white. 25The histological and DNA quantification results showed a significant reduction in the bone marrow elements, and the HE results showed normal cell morphology; Saf-O staining confirmed the high preservation of the GAG; and Sirius red staining demonstrated type I/III COL preservation in cartilage tissues in all groups (Figure 5).The residue (Figure S1.) in the 1.5-mL centrifuge tube was subjected to frozen section and HE staining and was found to contain large amounts of lipids, blood, and various cells (Figure S2).The chondrocyte viability results showed that HSC treatment for 2 min did not damage the chondrocytes (Figure 7).After zoning analysis of the subchondral bone was performed, and the histological and DNA quantification results showed that some bone marrow elements remained in the superficial zone of the subchondral bone after HSC, which the authors attributed to the absence of a dedicated HSC tube.Because the diameter of the OCAs was slightly smaller than that of the centrifuge tube, the subchondral bone clung to the wall of the tube, resulting in inadequate separation of the bone marrow in the superficial zone from the bone trabeculae.Moreover, the OCAs were centrifuged with a large amount of blood, lipids, and bone debris, which filled the space at the bottom of the tube, resulting in the bone marrow not being completely detached.To solve this problem, we designed a new centrifuge tube dedicated to centrifuging the OCAs to better remove the immunogenic elements of the subchondral bone and to apply larger OCAs.However, this study did not use the new tubes.
In studies relevant to the exploration of OCA failure and immune rejection, many methods have been proposed to suppress rejection, such as donor-host antigen match, pre-transplantation graft treatment methods, and posttransplantation rejection suppression methods, all of which may directly or indirectly promote bone healing and integration of the graft and host.A clinical study discovered that sex mismatch transplantation may lead to OCA failure, particularly when male grafts are transplanted into female donors, with significantly lower 5-year survival rates. 15Yang et al. 17 used basic fibroblast growth factor and agarose gel-coated OCAs implanted in homozygous rats to enhance immune privilege and showed that allografts and autologous transplantation produced the same healing effect.HSC could play an important role in decreasing immunogenicity and therefore has the potential to increase success.

Limitation and Strengths
This study has some limitations.First, porcine knee joints were selected instead of human tissues because porcine joint size, cartilage thickness, and biomechanics are similar to those of humans and have a strong history of use for OCAs preservation experiments. 20,25,35Additionally, porcine osteochondral bone is also the most likely source of xenografts. 42However, differences in bone density and porosity of subchondral bone between humans and pigs may impact the results of this study.Second, OCAs in this study were only 8.5 mm in diameter.The thickness and size of the grafts may have affected the experimental results.Finally, we did not explore the effects of various centrifugation parameters such as speed and duration.
Nonetheless, this study has the following strengths.First, chondrocytes and the extracellular matrix were not destroyed after using these five physical methods for removing immunogenic elements.Second, these methods can be performed aseptically in a sterile environment.Thus, these methods are potentially the method of choice in decreasing immunogenicity for pre-transplantation graft treatment.

Conclusion
I n summary, our study revealed that immunogenic ele- ments were not significantly reduced after saline, ultrasound, or VV treatments of OCAs, whereas there was a significant reduction in immunogenic elements after LPL, HPL, and HSC treatments.Furthermore, HSC more effectively reduced the immunogenic elements of OCAs than LPL and HPL; blood, lipid, and bone marrow elements in the subchondral bone decreased significantly after HSC treatment.Additionally, micro-CT, histological analyses, and chondrocyte viability results showed that HSC does not damage the subchondral bone and cartilage, whereas LPL and HPL may damage the subchondral bone.This study suggests that HSC has a potentially important role in decreasing immunogenicity and therefore may increase success in OCA transplantation.

FIGURE 1
FIGURE 1 Vertical section of the OCA.(A)Superficial (external third), middle (middle third), and deep (central third) zones identified on the fresh OCA.(B) Pathological sections of the OCA after HSC treatment.The black arrows point to the bone marrow cavity and the trabecular bone and bone marrow cells are shown in red.HSC, high-speed centrifugation; OCA, osteochondral allograft.

FIGURE 2
FIGURE 2 Weight variation of OCAs.Data are reported in box plots with mean, quartiles, and minimum and maximum values.Statistical analysis was performed using one-way analysis of variance comparing the control and each treatment group with Bonferroni's post-hoc test.*p < 0.05.HPL, high-pulse lavage; HSC, high-speed centrifugation; LPL, low-pulse lavage; OCAs, osteochondral allografts; VV, vortex vibration.

FIGURE 4 FIGURE 5
FIGURE 4 Gross photographs of OCAs after different method treatments.The trabeculae in the control group were all black.The peripheral trabeculae were yellow, and the central trabeculae were black in the saline, ultrasound, and VV groups.The peripheral trabeculae were white, and the central trabeculae were black in the LPL and HPL groups.The trabeculae in the HSC group were all white.HPL, high-pulse lavage; HSC, highspeed centrifugation; LPL, low-pulse lavage; OCAs, osteochondral allografts; VV, vortex vibration.

FIGURE 6
FIGURE 6 Evaluation of DNA concentration in different zones of OCAs.Data are reported in box plots with mean, quartiles, and minimum and maximum values.Statistical analysis was performed using one-way analysis of variance and the control and each treatment group were compared with Bonferroni's post-hoc test (*p < 0.05), and the LPL, HPL, and HSC groups were compared with Bonferroni's post-hoc test ( # p < 0.05).HPL, highpulse lavage; HSC, high-speed centrifugation; LPL, low-pulse lavage; OCAs, osteochondral allografts; VV, vortex vibration.

TABLE 1
Study groups and treatment methods In the superficial zone, a remarkable reduction of DNA concentration in the LPL, HPL, and HSC groups was observed in the control group, with mean values of 72.58, 63.48, and 95.23 ng/μL, respectively, compared to 205.85 ng/μL in fresh OCAs, reaching reductions of 64.74% (p = 0.0010), 69.16% (p = 0.0005), and 53.74% ( p = 0.0063).In the middle zone, a remarkable reduction of DNA concentration in the HSC groups was observed compared to the control group, with a mean of 76.48 ng/μL compared to 234.18 ng/μL in fresh OCAs, reaching 67.34% (p < 0.0001) reduction.Furthermore, the DNA concentration in the HSC group was lower than that in the LPL (p = 0.0009) and HPL (p = 0.

TABLE 2
Comparison of the pre-and post-treatment OCAs Micro-Computed Tomography Evaluation Note:The values are expressed as mean AE SD unless otherwise indicated.The p-value refers to the differences between the pre-treatment and post-treatment Micro-Computed Tomography Evaluation.; Abbreviations: BMD, bone mineral density; BV/TV, bone volume per tissue volume; Conn.D, connection density of bone trabeculae; HPL, high-pulse lavage; HSC, high-speed centrifugation; LPL, low-pulse lavage; OCAs, osteochondral allografts; Pre-T, Pre-treatment; Post-T, Post-treatment; Tb.N, trabecular number; Tb.Sp, trabecular space; VV, vortex vibration.*P < 0.05 was considered statistically significant.