The Effect of Pressurized Carbon Dioxide Lavage on Osteochondral Allograft

Abstract

Background:

Osteochondral allograft (OCA) transplantation is often utilized for treating osteochondral lesions of the knee due to limited cartilage regenerative capabilities. The use of liquid orthobiologic agents such as bone marrow aspirate concentrate has gained traction as a method to enhance OCA incorporation and healing. Pulse lavage and pressurized carbon dioxide (CO₂) lavage are cleaning methods used during OCA preparation to remove native marrow elements, but their isolated and combined effects are not fully understood.

Purpose/Hypothesis:

This study aimed to assess the impact of incorporating pressurized CO₂ lavage as an adjunct to pulse lavage during OCA preparation. It was hypothesized that adding pressurized CO₂ lavage would enhance the OCA absorption capacity for liquid-based materials such as host-derived marrow constituents.

Study Design:

Controlled laboratory study.

Methods:

OCA plugs were harvested from medial hemicondyles. The plugs were divided into 2 groups: one group underwent pulse lavage alone, and the other group underwent pulse lavage followed by pressurized CO₂ lavage (sequential group). The plugs were weighed before and after each cleaning step, and a water-based dye solution was added by dripping. Next, the liquid content was determined by centrifugation and spectrophotometry. Results were analyzed statistically.

Results:

A total of 34 OCA plugs with a 14 to 18 mm–diameter were harvested. After the cleaning phase, the sequential group showed a significant mean reduction in aqueous weight of 0.253 ± 0.110 g (107% ± 21%) (P < .0001) and a subsequent mean gain in aqueous weight of 0.231 ± 0.107 g (96% ± 19%) (P < .0001), after the cleaning and dye-dripping procedures. In contrast, the pulse lavage–only group exhibited a small mean weight gain of 0.028 ± 0.010 g (11% ± 4%) after the dye-dripping procedure.

Conclusion:

Adding pressurized CO₂ lavage to pulse lavage improved OCA cleaning and increased uptake of an aqueous dye solution used as a surrogate for liquid orthobiologic agents in this ex vivo model.

Clinical Relevance:

The study findings highlight the potential of incorporating pressurized CO₂ lavage as an adjunct to pulse lavage during OCA preparation. These findings could enhance OCA preparation techniques by improving the removal of donor marrow elements and facilitating the subsequent ingress of fluid into the graft's cancellous portion.

Keywords

osteochondral lesions osteochondral allograft transplantation pulse lavage pressurized carbon dioxide lavage

Cartilage lesions are commonly observed during knee arthroscopy and are prevalent among young and active patients.² When these lesions become symptomatic, they can lead to pain and functional limitations, and may progress to irreversible joint damage and osteoarthritis.^2,8,10,16 Because the articular cartilage has limited regenerative capacity, symptomatic focal defects often require surgical management.¹⁸ In a cohort of 993 consecutive knee arthroscopies, articular cartilage pathology was observed in approximately two-thirds of procedures, with localized defects reported in roughly 20% and localized full-thickness defects in approximately 11% of patients.² Various surgical procedures have been devised, with fresh osteochondral allograft (OCA) transplantation surgery typically reserved for significant defects that exceed 2 to 4 cm² and involve the subchondral bone. Additionally, it may be considered in cases in which previous attempts of cartilage surgery have been unsuccessful.^9,16,18 During the surgical procedure, a suitable cylindrical plug (OCA) is selected from a donor graft to match the defect and is then secured in place either by press-fitting or with interfragmentary screws.⁹

Successful graft incorporation depends largely on osseous integration of the subchondral bone portion of the allograft and on minimizing residual marrow elements that may contribute to an immunologic response.^18,19,26 Additionally, in clinical practice, the osseous portion of the OCA plug is often exposed to liquid orthobiologic preparations, such as bone marrow aspirate concentrate, to deliver bioactive cells and growth factors to the graft interface, potentially facilitating early osseous incorporation.^11,13,15,17 However, the available clinical evidence supporting orthobiologic augmentation remains mixed, with some studies reporting improved early radiographic integration and others showing no clear benefit on advanced imaging or longer-term follow-up.^11,27,31

Pulse lavage is commonly used during OCA preparation to remove residual bone marrow, fat, and blood products from the cancellous portion of the graft.^1,26,30 Residual marrow elements have been linked to the immunologic response to OCA, prompting evaluations of lavage techniques to mitigate these immunogenic components.^19,25,26 The amount of marrow removed by pulse lavage is variable and depends on lavage duration and flow intensity.^1,30 Pressurized carbon dioxide (CO₂) lavage has been evaluated as an adjunct method to further clean cancellous bone surfaces and improve cement penetration in arthroplasty studies.^5,23,24 It has also been marketed and recommended for bone preparation, including OCA preparation.²² More effective marrow removal may increase available pore space within the cancellous bone, which could facilitate the ingress of host-derived cells and fluids after implantation, including locally derived marrow constituents or applied liquid adjuncts.^3,26

This study aimed to assess the impact of incorporating pressurized CO₂ lavage as an adjunct to pulse lavage during OCA preparation. We hypothesized that adding pressurized CO₂ lavage would create extra space and enhance the OCA absorption capacity for liquid-based materials such as host-derived marrow constituents.

Methods

After the supplier's self-imposed 28-day expiration period, 9 fresh-frozen medial femoral hemicondyles were donated by RTI Surgical and stored at −80°C until needed. The hemicondyles were thawed 24 hours before plug harvest. Before harvesting, each hemicondyle was measured using a template sizer to determine the possible plug diameters that could be extracted from the specimen. Four plugs, each with a diameter ranging from 14 to 18 mm, were templated to each hemi-condyle. Sizing was conducted to facilitate the planned harvest sequence while ensuring that subsequent plug sites remained undisturbed.

The plugs were divided into 2 groups: the first group received only pulse lavage, while the second group, referred to as the sequential group, underwent a cleaning process involving pulse lavage followed by pressurized CO₂ lavage. A fellowship-trained orthopaedic surgeon (S.L.S.) performed the harvest and OCA preparation process. Per our institution's guidelines, studies involving tissue of anonymous donors obtained from accredited tissue banks for research purposes are exempt from institutional review board approval.

OCA Harvest

The OCA plugs were harvested using designated sets, specifically the Arthrex Allograft OATS Set (large) and the RTI Surgical Precision Allograft Cartilage Kit. The hemicondyle was firmly secured in a vise to achieve the desired orientation, with the articulation arm adjusted to align with the contour of the hemicondyle and positioned perpendicularly (Figure 1A). Using the designated reamer, we extracted the first plug from the most posterior aspect of the hemicondyle, moving toward the most anterior part. Throughout this process, a guide wire was used to ensure that the reaming path would not interfere with the placement of the subsequent plug. The harvesting process was then conducted using a sagittal saw positioned perpendicular to the plug at the approximate depth reached during reaming (Figure 1B). Once all 4 plugs were successfully harvested, they were marked at 8-mm depths from the cartilage surface and placed into the sizing block (Figure 1C). The plugs were secured and then finished by cutting each to the 8-mm depth.

Figure 1.

(A) The hemicondyle clamped in a vise, with the reamer positioned within the articulation arm, adjusted perpendicularly to align with the contour of the hemicondyle. (B) Utilization of the sagittal saw for the plug harvest. (C) An 8 mm–depth mark from the surface of the cartilage (indicated by a dashed blue line).

OCA Preparation

Phase 1

The OCA plugs were weighed and cleaned using 2 cleaning methods (Figure 2A). First, all the plugs were cleaned using a pulse lavage system (Stryker). To ensure consistency, the tip of the lavage was positioned within 5 mm of the OCA plug, and each plug was thoroughly rinsed with 1 L of saline solution (Figure 2B). Subsequently, the plugs were weighed again and divided into 2 groups. The first group was solely treated with the pulse lavage, while the second group (ie, sequential group) underwent additional cleaning using CO₂ lavage administered by a CarboJet device (Kinamed). Per the manufacturer's recommendation,^21,22 the CarboJet CO₂ wall pressure was set to 344 kPa (50 PSI), and the CarboJet nozzle's handpiece was positioned no more than 2 mm from the surface of the plug for 1 minute (Figure 2C). Subsequently, all the plugs underwent another weight measurement and were left to air-dry for 15 minutes, with the cartilage surface facing downward.

Figure 2.

(A) Schematic flowchart of phase 1 featuring the harvested plug positioned with the cartilage surface facing downward. (B) Pulse lavage cleaning. (C) Carbon dioxide (CarboJet device) cleaning. (D) Application of dye dripping utilizing a 2-mL solution of 440 μM resazurin.

To simulate the application of an orthobiologic agent to the OCA plugs, a 2-mL solution of 440 μM resazurin dye (Sigma-Aldrich) in phosphate-buffered saline was carefully applied to the bony surface to ensure maximum absorption. Any excess dye was drained, and the plugs were reweighed (Figure 2D). Resazurin (Alamar Blue) was selected for its well-established use in spectrophotometric assays, minimal absorption by tissue compared to conventional laboratory dyes, and high solubility in aqueous buffers.⁶ These properties enabled accurate quantification, reduced dye loss after bone contact, and allowed for clear phase separation from organic debris. To establish and validate the protocol, 2 initial plugs were used for preliminary testing of the centrifugation protocol and were subsequently excluded from the analysis.

Phase 2

To determine the amount of dye absorbed, the bone plugs were subjected to centrifugation to separate the liquid from the plugs (Figure 3A). The centrifuge was set to 300g (also referred to as relative centrifugal force) for 10 minutes. The time for centrifugation was established through a preliminary assessment, which involved a gradual increment in the centrifugation time until a weight loss of <1% was observed. After the centrifugation process (Figure 3B), the resulting residue (referred to as the “total liquid”) displayed 2 distinct layers. The upper layer, referred to as the “organic layer,” primarily consisted of bone components and fat derived from the bone marrow. The lower layer, referred to as the “aqueous layer,” consisted of the dye and any remaining saline solution. Next, the total liquid containing both layers was weighed along with the specific measurement of the aqueous layer (Figure 3C). The latter was then subjected to analysis by a spectrophotometer (also referred to as a plate reader) to determine the concentration of the dye with an established standard curve. By comparing this concentration to the original dye concentration, we were able to determine the volume/weight of the original dye solution retained by each plug. Lastly, the centrifuged graft was bisected to evaluate the presence of any remaining dye (Figure 3). This step was used only to check for visible pooling of dye solution after centrifugation; it was not used to quantify uptake or compare groups. Depth of wash penetration was indirectly assessed by calculating the proportion of residual organic material (pale-yellow layer) separated from the resazurin solution after centrifugation. A lower percentage of this organic fraction relative to the total recovered liquid was used as an index of deeper penetration.

Figure 3.

Schematic flowchart of phase 2 illustrating the centrifugation process. (A) The harvested bone plug after dye dripping. (B) The OCA plug after centrifugation. (C) Measuring the weight of the plugs using an electronic weight scale. (D) The OCA plug after centrifugation cut in half. Numbers 1 and 2 represent the pulse lavage–only group and the sequential group, respectively. Note: Images are qualitative only; for quantitative analysis, spectrophotometry of the recovered aqueous layer with a standard curve was used.

Statistical Analysis

Statistical analysis was carried out using SAS Version 9.4 software (SAS Institute). All data are presented as mean ± standard deviation. A P value <.05 was considered significant. For the comparison between the 2 groups, we used the Student t test. Pearson correlation coefficient analysis was used to assess the strength of the relationship between the original weight of the OCA plugs and the weight of the liquid obtained from the centrifugation step. A Pearson correlation coefficient value of −1 indicates a complete negative linear correlation, a value of 0 represents no correlation, and a value of +1 signifies a complete positive correlation.

Results

Nine freshly stored medial hemicondyles were used to obtain OCA plugs, resulting in 34 osteochondral grafts for analysis. Half of the samples underwent pulse lavage alone, while the other half underwent sequential pulse lavage followed by pressurized CO₂ lavage (Table 1).

Table 1

Sample Number and OCA Plug Size in Each Cleaning Procedure ^a

Cleaning Procedure	No. of Condyles	No. of 8 mm–Thickness Plugs	Diameter (mm)			Mean ± SD (mm) ^c
Cleaning Procedure	No. of Condyles	No. of 8 mm–Thickness Plugs	18	16	14	Mean ± SD (mm) ^c
Pulse lavage	9	17	10	3	4	16.71 ± 1.72
Sequential ^b	9	17	7	5	5	16.24 ± 1.71

OCA, osteochondral allograft.

Sequential refers to pulse lavage followed by CO₂ lavage cleaning.

P = .53.

When examining the size and weight changes of the grafts, it was observed that the sequential procedure had a higher proportion of smaller plugs (<18 mm) compared to the lavage-only group. However, this difference was not statistically significant (P = .61). As a result, the mean weight of the initial plugs in the sequential group was lower. During phase 1, both groups had a minimal weight change after pulse lavage cleaning (Table 2).

Table 2

Mean Plug Weight and Liquid Weight (g) at Each Step ^a

	Pulse Lavage	Sequential	P Value
Phase 1
Initial plug weight	4.26 ± 0.76	4.12 ± 0.82	.61
Postlavage	4.27 ± 0.76	4.12 ± 0.82	.59
Post–CO₂ lavage	NA ^b	4.07 ± 0.83	NA ^b
Postdripping	4.30 ± 0.77	4.09 ± 0.82	.47
Phase 2
Total liquid	0.46 ± 0.19	0.37 ± 0.14	.15
Aqueous	0.28 ± 0.11	0.25 ± 0.09	.51
Dripped dye ^c	0.13 ± 0.07	0.17 ± 0.08	.11

NA, not applicable.

This group did not undergo CarboJet cleaning.

Dripped dye refers to the amount of dye solution (defined as 100% concentration for spectrophotometric analysis) retained within the plugs after application.

However, after the CO₂ lavage, the plugs underwent a mean weight loss of 0.05 g, which was partially regained after the dripping step. In phase 2, the total liquid accounted for approximately 16% of the initial plug weight, with the weight of the dye that was dripped onto the plug being lower than the total liquid retained (Table 2). Similar results were noted when analyzing the weight changes during phase 1, manifesting as liquid weight variations at each step. This change was determined by subtracting the plug weight at the current step from the plug weight of the previous step (Table 3).

Table 3

Mean Weight Change (g) at Each Step of Phase 1 Study ^a

	Pulse Lavage	Sequential
Postlavage	0.0004 ± 0.0193	0.0026 ± 0.0196
Post–CO₂ lavage	NA	–0.2527 ± 0.1066
Postdripping	0.0282 ± 0.0097	0.2308 ± 0.1071

Negative values indicate weight loss, whereas positive values indicate weight gain. NA, not applicable.

Because the sizes and weights of plugs for each procedure differed, a baseline normalization was necessary to ensure precise comparisons. On conducting Pearson correlation analysis, it was found that the initial plug weight and total liquid weight were independent factors with no significant correlation (coefficient, 0.21781; P = .1247), while the aqueous liquid weight was highly correlated to the total liquid weight (coefficient, 0.87808; P < .0001). As the primary objective of this study was to assess the impact of cleaning procedures on liquid content, we selected the aqueous liquid weight for each plug as the baseline for normalization. Consequently, the data were presented as a percentage of the aqueous liquid content (Figure 4A). Compared to the aqueous liquid content of the plugs, we observed a significant weight reduction of 107% ± 21% (P < .0001) in the sequential group after the implementation of CO₂ lavage. Additionally, there was a significant weight increase of 96% ± 19% (P < .0001) after adding dye through the dripping process. In contrast, the pulse lavage–only group exhibited minimal weight loss and an insignificant weight gain of 11% ± 4% after the dye-dripping procedure. Finally, the results of the analysis of the color of the aqueous liquid obtained from the postdripping plugs, which reflects the initial dye content, can be found in Figure 4. This process emulates the quantity of retained liquid orthobiologics for each technique; the goal is to simulate the quantity of retained liquid orthobiologics using each respective method (Figure 4B). The sequential group still demonstrated a greater absorption capacity compared to the pulse lavage–only group; however, both groups exhibited a significant increase in dye absorption, with increments of 67% ± 11% and 47% ± 10%, respectively.

Figure 4.

(A) The relative weight changes in each group normalized to the aqueous liquid weight. The data are presented as a percentage of the overall liquid content. Each stage is demarcated by a dashed line, with the asterisk denoting statistical significance (P < .0001). (B) The color of the aqueous liquid collected from postdripping plugs by centrifugation (P < .0001).

The mean penetration index was lower in the sequential wash group (29.08% ± 16.66%) compared to the pulse lavage group (37.92% ± 12.19%), suggesting deeper penetration; however, the difference did not reach statistical significance (P = .0872). These findings are further supported by the absence of residual dye solution observed in the bisected graft after centrifugation (Figure 3D). As the resazurin oxidation-reduction dye is exposed to air/oxygen for an extended period, it eventually turns from blue to red.

The absence of visible pooled dye solution in the bisected grafts after centrifugation, as shown in Figure 3D, further supports these findings. Since visual color intensity can be affected by retained aqueous fluid, dilution, and the handling of resazurin, it was not used for comparisons between groups. Instead, the quantitative assessment of simulated orthobiologic uptake was based on spectrophotometric analysis of the recovered aqueous layer, as depicted in Figure 4B.

Discussion

The objective of this study was to evaluate how cleaning procedures affect the absorption ability and liquid content of OCA plugs. To achieve this, we compared the effects of pulse lavage alone with a combination of pulse lavage and CO₂ lavage. We intended to replicate the surgical procedure of incorporating an orthobiologic agent such as concentrated bone marrow aspirate (cBMA) to promote the integration and healing of OCA. The study's primary outcomes revealed that adding CO₂ lavage to pulse lavage improved the cleaning process by generating more vacant space and considerably enhancing the plug's absorption capacity.

Prior research on pulse lavage primarily concentrated on its efficacy in eliminating bone marrow elements and blood clots, as well as reducing donor immunogenicity.^{1,19,23,26,28,31} Sun and colleagues³⁰ investigated OCA cores that were subjected to different durations, flow rates, and intensities of pulse lavage to assess the impact on bone marrow cleaning. They concluded that pulse lavage can effectively clean the bone marrow, and its cleansing effectiveness is closely associated with these factors, such as time and pressure.³⁰ Numerous other studies support this conclusion, demonstrating the effectiveness of pulse lavage in cleaning cancellous bone.^7,12,20 On the other hand, Ambra et al¹ evaluated 48 fresh OCA plugs and analyzed their DNA content after undergoing pulse lavage cleaning. They discovered that pulse lavage had limited effectiveness in fully removing marrow elements, with lower efficacy in larger-diameter and thicker depth plugs.

The application of CO₂ lavage has been predominantly studied in knee arthroplasty procedures, which demand meticulous bone preparation to enhance the bone-cement interface and facilitate cement penetration into the cancellous bone.^5,22-24 Knappe et al²⁴ conducted a laboratory study comparing the impact pressure and cleaning effects of pulsatile saline lavage and CO₂ lavage on cancellous bone. The researchers found that the combination of both techniques led to greater cleaning effect compared to each technique separately.²³ However, this effect did not reach statistical significance. In a separate cadaveric study conducted by the same research team to evaluate the cement penetration and stability of the bone-cement interface for the knee arthroplasty procedure, it was found that the supplementary use of CO₂ lavage with high-pressure pulsatile saline lavage did not provide any additional advantage.²³

On the contrary, in their retrospective study, Gapinski et al¹⁴ reviewed 303 primary total knee arthroplasty surgeries performed with and without a tourniquet, along with the addition of CO₂ lavage after a thorough pulsatile lavage cleaning. Their findings revealed that bone prepared with CO₂ lavage exhibited significantly higher levels of cement penetration compared to bone prepared solely with pulsatile lavage, regardless of whether a tourniquet was used or not. Baumann and colleagues³ explored different techniques for OCA preparation including saline and CO₂ treatment before adding cBMA. They found that cleaning with CO₂ reduced colony-forming units, improved marrow element removal, and improved osteoinductive biomarkers from cBMA.³

Other studies examining the combined impact of pressurized CO₂ lavage and pulse lavage have also arrived at the same conclusion regarding their beneficial effects.^5,28 In their study, Meyer et al²⁶ compared the efficacy of saline lavage with a combination of saline and high-pressure CO₂ lavage in the removal of marrow elements from various depths within fresh OCAs. The study incorporated 3 treatment groups: no lavage, pulse lavage only, and saline lavage followed by CO₂ lavage. While both lavage methods effectively removed marrow elements from the superficial and middle zones, the addition of CO₂ lavage resulted in significantly more removal of marrow elements from the deep zone. Consequently, the authors concluded that the combination of saline and high-pressure CO₂ lavage is more efficient in clearing marrow elements from OCAs compared to saline lavage alone.

Similarly, the findings of this study demonstrate that CO₂ lavage improved the ability of OCA plugs to absorb liquid agents such as cBMA, as indicated by changes in weight and liquid content. When examining the pulse lavage–only group during phase 1 (Table 3), a negligible weight loss of 0.0004 ± 0.0193 g was observed after cleaning, followed by an insignificant weight gain of 0.0282 ± 0.0097 g after dye dripping. In contrast, the sequential cleaning process resulted in a notable weight loss and subsequent weight gain of – 0.2527 ± 0.1066 g and 0.2308 ± 0.1071 g, respectively, after the utilization of CO₂ lavage. In phase 2, a similar pattern was observed, but with even more pronounced effects when using the aqueous liquid weight as the baseline for normalization. Compared to the pulse lavage–only group, which exhibited insignificant weight changes of 11% after the dye-dripping procedure, the sequential group with CO₂ lavage cleaning displayed significant weight changes exceeding 96% (P < .0001). This represents a 9-fold increase compared to the pulse lavage–only group. These findings suggest that CO₂ lavage more effectively removes residual liquid from the plug, subsequently allowing greater dye absorption. Finally, when evaluating the amount of dye retained by the plugs, both groups exhibited a significant increase, with a 47% increase in the pulse lavage–only group compared to a 67% increase in the sequential group. A possible explanation for the observed insignificant weight changes in the pulse lavage–only group was proposed in a study by Ambra et al.¹ The authors speculated that pulse lavage might have the tendency to concentrate bone marrow elements in the center of the plug instead of effectively removing them from the plug altogether. Moreover, it is important to note that in the current study, neither of the cleaning processes demonstrated complete removal of all liquid from the plugs, as evidenced in Table 2, where the weight of the retained dye after dripping was lower than the total liquid weight. The presence of the organic layer in the total liquid after centrifugation (Figure 3) further reinforces this finding. If the pulse lavage or CO₂ lavage could effectively remove all the liquid from the plug, the absence of an organic layer postcentrifugation would be expected. This discrepancy could potentially be attributed to the manner in which the plugs are dried subsequent to rinsing.

In the current study, unlike prior studies, liquid orthobiologic agent absorption during OCA transplantation was simulated by dripping 2 mL of dye solution onto the plugs after cleaning using the 2 techniques. Since the mean weights of the plugs’ aqueous solution in the pulse lavage and sequential groups were 0.278 and 0.254 g, respectively, the amount of excessive 2-mL dye solution used in this study was about 7.19- to 7.87-fold higher than the available space in plugs. As depicted in Figure 4A, the dripped dye occupied these vacant areas, but with a significant weight difference of >9 times between the pulse lavage–only and sequential groups after the dye-dripping process. Another purpose of the dye solution was to measure the dye content retained inside the plugs after dripping. Since the excess dye solution does not significantly affect the weight change after dripping, as any excess solution is removed before weighing, it does affect the amount of dye content retained in the plugs, resulting in a notable but reduced difference between the 2 cleaning groups (Figure 4B).

In the pulse lavage–only group, there was a significant 47% increase in dye content as the excess dye solution (100% concentration) was dripped into the saline-filled space (0% concentration) in a continuous manner. This gradual increase in dye concentration led to higher retention of dye in the plugs. On the other hand, the sequential group plugs showed a mean dye content of 67%. We attribute these lower than expected results in the sequential group to the ample empty space available, which allows a large amount of dye solution to access the plugs from the dripping. This continuous supply of excessive dye solution leads to a greater dye absorption by the bone structure, resulting in a reduced dye content when measuring the pulled-out solution through centrifugation. The same concept applies to the results of the pulse lavage–only group, but there are notable differences. The pulse lavage–only group starts with a lower initial dye content, and the exposure time to a higher dye content is relatively shorter. These factors may contribute to a lower measured dye content in the pulse lavage–only group compared to the sequential group. Figure 3D is included solely for qualitative illustration, as visual color differences may be affected by retained aqueous fluid and dilution and were not used for comparisons between groups. In addition, because an aqueous dye solution was used as a surrogate, cellular components within biologic agents such as cBMA may behave differently than dye molecules within cancellous bone.

In contrast to previous studies that primarily examined the removal of bone marrow elements and cement penetration as a measure of cleaning effectiveness,^{1,5,7,14,23,24,30,31} this study specifically focused on the creation of additional space and the enhanced absorption capacity of OCA plugs after the combined application of pulse lavage and CO₂ lavage. Apart from the cleaning aspect, the additional space and improved absorption capacity may have the potential to increase the volume of absorbed orthobiologic agents such as cBMA, which in turn can facilitate improved integration of the OCA and promote accelerated healing processes.

Limitations

There are several limitations to this study. First, the grafts used in the study exhibited variations in size and shape due to technical and anatomic considerations. Although all OCA plugs were normalized and presented as percentages, these differences in size may have influenced the cleaning capability.³⁰ Similarly, a consistent depth of 8 mm was used for each graft. There is potential variability in the penetration and clearing of contents with different depths for each method. The time spent cleaning the grafts can be modified to remove further contents.³⁰ Additionally, using the air-drying method for the OCA plugs may have left residual liquid in the grafts, which could have interfered with the results. Furthermore, the grafts were stored in the freezer at −80°C and thawed 24 hours before use, potentially impacting the cleaning process and subsequent results.^9,30 Finally, instead of utilizing an orthobiologic agent, a 2-mL water-based solution of 440 μM resazurin dye was used in this study. This solution differs in composition from liquid orthobiologic agents such as cBMA, which contains bone marrow elements and is more lipid-rich than a water-based solution.²⁹ Visual color differences in bisected plugs are qualitative and may be influenced by retained aqueous fluid and dilution; therefore, color was not used for between-group comparisons. Quantitative comparisons relied on spectrophotometry of the recovered aqueous layer using a standard curve, as described in the Methods section.

Regarding the use of CO₂ lavage, there are additional limitations to note. CO₂ lavage is not a ubiquitous practice, and while all operating rooms should have access to the gas, the ability to use it for procedures may be limited at some institutions. While the additional step of cleaning the OCA plugs with CO₂ does extend the procedure time, our experience suggests this extension is minimal, yet it remains a consideration for surgeons. Additionally, the cost of the CarboJet device, which one study reported as $130 per use at their institution, must be taken into account.¹⁴ While studies have found CO₂ lavage to be sterile and safe, studies confirming that it is not harmful to OCA plug infrastructure are still needed.⁴

Conclusion

This study found that the combination of pulse lavage and pressurized CO₂ lavage in OCA preparation leads to a significant increase in available space and absorption capacity for liquid material agents such as cBMA. These findings suggest that both cleaning methods could be used before the addition of liquid orthobiologic agents for improved absorption of these agents. This could potentially help with optimal OCA implantation. However, it is recommended to conduct further clinical studies to reinforce and validate these conclusions.

Footnotes

Final revision submitted February 15, 2026; accepted March 2, 2026.

One or more of the authors has declared the following potential conflict of interest or source of funding: S.L.S is on the board of directors for ISAKOS and the ACL Study Group; holds committee positions for AAOS, AOSSM, AANA, ISAKOS, ICRS, and the Biologics Alliance; is an editorial board member for Orthopedics Today, Current Reviews in Musculoskeletal Medicine, and VJSM; is a course co-chair of ISMF, OSET Knee Sports, AO Sports, and the PFF Masters Course; is a paid educational consultant for Arthrex, LifeNet, Smith & Nephew, and Vericel; is an advisory board member for Kyniska Robotics, Reparel, Sarcio, Sparta Biomedical, Vericel, and Vivorte; receives royalties from ConMed and DJO; serves on a brace design team for DJO; holds stock or stock options for Arcuro Medical, Epicrispr BioTech, Icarus Medical, Kyniska Robotics, Moximed, Reparel, Sarcio, Sparta Biomedical, and Vivorte; and receives research support from AlloSource, JRF, Kinamed, Miach Orthopaedics, Octane Biotherapeutics, Organogenesis, Ossio, and University of Pittsburgh. B.J.C. has received research support from Aesculap/B.Braun, Arthrex, and National Institutes of Health (National Institute of Arthritis and Musculoskeletal and Skin Diseases and National Institute of Child Health and Human Development); is a paid consultant for Acumed, Arthrex, Bioventus, DJO, Endo Pharmaceuticals, Flexion Therapeutics, Geistlich Pharma, North America, Pacira Pharmaceuticals, and Vericel Corporation; is an editorial or governing board member of the American Journal of Sports Medicine and Journal of the American Academy of Orthopaedic Surgeons; is a board or committee member of the Arthroscopy Association of North America; has stock or stock options in Bandgrip and Ossio; receives IP royalties from Arthrex and Elsevier Publishing; has received hospitality payments from Encore Medical LP, Flexion Therapeutics, GE Healthcare, GeistlichPharma, North America, Mallinckrodt, Merck Shapre and Dohme Corporation, Organogenesis, Orthofix Medical, PAVmed, Pinnacle, Pylant Medical, Summit Surgical Corp, Trice Medical, and Zimmer Biomet Holdings; has received other financial or material support from JRF Ortho; is a paid presenter for LifeNet Health and Terumo BCT; has received publishing royalties and financial or material support from Operative Techniques in Sports Medicine; and is a paid consultant for Ferring Pharmaceuticals. R.A. has received education payments from Arthrex and Smith & Nephew. K.L. has received education payments from Arthrex and Smith & Nephew. G.A. has received education payments from Arthrex and consulting fees from Bioventus.

ORCID iDs

Wen-Teh Chang

Stewart A. Bryant

Allen Seo

Brian J. Cole

Seth L. Sherman

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