Current Techniques for Processing Osteochondral Allografts Are Variable: A Systematic Review

Abstract

Purpose

To investigate the current optimal processing and storage protocols for fresh cold-stored osteochondral allografts (OCAs) used for resurfacing osteochondral defects in the knee.

Methods

Using the Preferred Reporting Items for Systematic Review and Meta-Analyses guidelines, 3 databases (PubMed, Embase, and Scopus) were queried for peer-reviewed articles on OCA storage and processing techniques. Articles were excluded if performed on nonhuman subjects or were narrative or systematic reviews, meta-analyses, or other forms of secondary sources. In vivo data included study type, population size, follow-up time, type, site and location of injury, description of surgery, description of postoperative rehabilitation, and outcome criteria used. Ex vivo data collection included tissue source and type, storage procedure including temperature, media changes, gas environment, storage solution including type of solution and additive used, cell viability methodology, and outcomes methodology.

Results

In total, 386 studies were screened between the 3 databases, with 27 studies satisfying all criteria. Eight human studies were included, with mean Modified Coleman Methodology Score of 48.38 ± 5.73. In total, 397 total patients were included with a mean of 49.6 patients per study. Five of the 8 studies had follow-up greater than 24 months. Three articles were retrospective studies, 4 were case series, 1 was a prospective cohort. Four articles were Level III evidence, 4 articles were Level IV evidence. In addition, 19 ex vivo human studies were included. A total of 78.95% of studies included grafts stored at 4°C or 1 to 10°C, 31.58% investigated 37°C, and 21.05% investigated room temperature. In total, 19 different storage media were investigated, with 68.42% including various additives.

Conclusions

OCA storage at 4°C remains the most common temperature with the most evidence-based research. However, investigation of OCAs at 37°C and room temperature, particularly those stored with proprietary protocol such as the Missouri Osteochondral Preservation System, have shown promising results at improved maintenance of viable chondrocyte density. Variability in storage media remains without clear consensus.

Clinical Relevance

A variety of methodologies are used for OCAs, and the best strategies are not well understood. There is a need to compile the available evidence from in vivo and ex vivo studies of OCAs to resolve conflicts regarding various available methodologies and provide better understanding of current techniques.

Articular cartilage defects affect a large number of patients and have limited healing potential. When left untreated, they can progress to osteoarthritis, which affects 46 million Americans older than age 25 years, accounting for annual expenditures of more than $185 billion dollars to the American health care system, as well as causing debilitating pain and adverse impact on functional quality.1, 2, 3, 4, 5

In younger patients, in whom the indications for arthroplasty may not be an ideal treatment option, cartilage resurfacing with an osteochondral allograft (OCA) has proven to be an effective option for joint preservation and has currently evolved into a more common standard of care for treating large (>2-4 cm²) full-thickness osteochondral defects that fail primary treatment regimens or as primary treatment in certain indications.^6,7 OCAs provide viable chondrocytes and mature hyaline cartilage while also restoring compromised underlying bone stock. They are a safe and effective option for the treatment of osteochondral lesions, posttraumatic arthritis, osteonecrosis, failed fixation of osteochondritis dissecans lesions, and other failed primary cartilage restoration procedures, including debridement and various marrow stimulation procedures.^6,8,9

Over the years, research into the impact of different storage parameters on the shelf life and quality of OCAs has substantially increased. For example, studies have shown the use of fresh OCAs transplanted within days to have shown clinical success.^6,8,9 Despite this growing body of work, the link between storage methods and clinical outcomes remains unclear. Studies have shown links between specific storage techniques, including tissue type, tissue storage, and media formulation with positive outcomes.^6,8,9 However, there is little clinical consensus on optimal protocols for storage. Of difficulty is the variability in the clinical factors that influence outcomes, including a wide array of tissue sources, grafts, media, and environmental conditions, that are continuously being introduced and evolving.

The purpose of this systematic review was to investigate the current optimal processing and storage protocols for fresh cold-stored OCAs used for resurfacing osteochondral defects in the knee. Our hypothesis was that optimal OCA-processing protocols would not be widely in use today but could be discovered through systematic literature review.

Methods

Identification

Using the Preferred Reporting Items for Systematic Review and Meta-Analyses guidelines, 3 databases (PubMed, Embase, and Scopus) were queried by 2 independent reviewers (M.M. and K.P.) for peer-reviewed articles on OCA storage (Fig 1). Discrepancies were discussed and resolved between the 2 reviewers. The databases were queried in January 2024 using the search terms “osteochondral allograft storage.” The time frame for the search included all articles up until January 2024.

Fig 1.

Preferred Reporting Items for Systematic Reviews and Meta-Analyses study algorithm used.

Inclusion and Exclusion

Articles were included if they were human knee tissue ex vivo or in vivo studies. Ex vivo inclusion criteria included articles that studies human cartilage research outcomes after tissue explant storage. In vivo inclusion criteria included studies in humans that investigated osteochondral cartilage health before implantation of allografts as well as osteochondral health repair outcomes of fresh OCAs. Articles with both knee and another joint OCA were included only if outcome measurements were described independently for each joint. Articles were excluded if they were performed on nonhuman subjects, were duplicates from previous databases, were not written in English, or were narrative reviews, systematic reviews, meta-analyses, or other forms of secondary sources. Articles were first screened by title, then by abstract content, then by full-text review.

Data Collection

To appropriately extract pertinent information from the identified studies, 2 separate standardized Excel datasheets (Microsoft, Redmond, WA) were developed for in vivo and ex vivo study types. In vivo data collection included study type, population size, follow-up time, type, site and location of injury, description of surgery if included, description of postoperative rehabilitation if included, and outcome criteria used. Ex vivo data collection included tissue source and type, storage procedure including temperature, media changes, gas environment, storage solution including type of solution and additive used, cell viability methodology, and outcomes methodology.

Data Analysis

Articles were further separated on the basis of whether they were human clinical or ex vivo studies. Human clinical papers were evaluated for study design using a modified Coleman Methodology Score (Table 1).¹⁰ Coleman’s methodology score uses 10 criteria to produce a score between 0 and 100, with 100 indicating a study that avoids confounders and biases.¹⁰ The 10 criteria that comprise Coleman’s methodology include population size, mean follow-up time, number of surgeries per reported outcome, study type, type and location of injury, surgical description given, rehabilitation description given, outcome criteria, procedure for how patients were recruited, and description of selection process for patients. The score assesses several key components including study design, which evaluates whether the study is randomized, controlled, prospective, or retrospective; inclusion and exclusion criteria, ensuring the study population is well-defined; sample size and power, which gauges if the study has enough participants to detect meaningful differences; and outcome measures, examining if valid, reliable, and relevant tools are used to measure results. Coleman’s methodology was used because it has been shown to be a validated scoring criteria that considers the study design and the quality outcomes that the study reports.^10,11 Descriptive statistics and a Pearson correlation comparing Coleman Methodology Score and publication date were obtained (Table 1). Ex vivo studies were evaluated on the basis of a methodology modified from Tabbaa et al.¹² that included tissue source and type, storage procedure including temperature, media changes, gas environment, storage solution including type of solution and addictive used, cell viability methodology, and outcomes methodology. Descriptive statistics for each criterion were obtained.

Table 1.

Modified Coleman Methodology Score¹⁰

	Score
Part A. Only one score to be given for each section
Study size: patient number, n
>60	10
41-60	7
20-40	4
<20, no stated	0
Mean follow-up, mo
>24	5
12-24	2
<12	0
Number of different surgical sites included in each reported outcome
One surgical site	10
More than 1 surgical site, but >90% of subjects undergoing 1 site, <10 concomitant site	7
Not stated, unclear, or <90% of subjects undergoing the 1 site	0
Type of study
Randomized controlled trial	15
Prospective cohort study	10
Retrospective cohort study	0
Diagnostic certainty (type and location of lesion described)
Location and type of lesion described	5
Location or type of lesion described	3
None described	0
Description of surgical procedure given
Adequate	5
Fair	3
Inadequate, not stated, or unclear	0
Description of postoperative rehabilitation
Well described	10
Inadequate, not stated, or unclear	0
Part B. Scores may be given for each option in each of the 3 sections if applicable
Outcome criteria
Outcomes measure clearly defined	2
Timing of outcome assessment clearly stated	2
Use of outcome criteria that has reported good reliability	3
Use if outcome with good sensitivity	3
Procedure for assessing outcomes
Subjects recruited (results not taken from surgeons' files)	5
Investigator independent of surgeon	4
Written assessment	3
Completion of assessment of subjects themselves with minimal investigator assistance	3
Description of subject selection process
Selection criteria reported and unbiased	5
Recruitment rate reported >80%	5
Recruitment rate reported <80%	3
Eligible subjects not included in the study satisfactory accounted for or 100% recruitment	5
Total score

NOTE. Shown is the Modified Coleman Methodology scoring breakdown used for human clinical studies.¹⁰

Results

Study Characteristics

The initial search of the 3 databases resulted in 386 initial studies. After 3 rounds of screening by 2 independent reviewers on the basis of title, abstract, and full-text review, a result of 27 studies satisfied all inclusion and exclusion criteria. Eight studies were human clinical research, whereas 19 studies were human ex vivo research. The ex vivo research encompassed studies that investigated storage conditions without recipient transplantation for human OCAs that were either obtained from surgeries or human cadaveric donors.

Human Clinical Studies

Eight human clinical papers were included in the study. The overall mean Modified Coleman Methodology Score was 48.38 ± 5.73 (Table 2).13, 14, 15, 16, 17, 18, 19, 20 There was a weak correlation found between the year of publication and methodology score (r = 0.094). Only one study, by Williams et al.,⁶ was published more than 10 years ago.

Table 2.

Modified Coleman Methodology Score for Clinical Human Studies

Title	Rauck et al., 201913	Stoker et al., 201814	Schmidt et al., 201715	Tírico et al., 20167	Williams et al., 20076	Nuelle et al., 201716	Merkely et al., 202017	Cinats et al., 202118	Average
Part A. Only one score to be given for each section
Study size – number of patients (N)	0	10	10	0	0	10	10	0	5
Mean follow-up	0	0	5	5	5	2	5	5	3.375
Number of different surgical procedures included in each reported outcome.	10	10	10	10	10	10	10	10	10
Type of study	0	10	0	0	0	0	0	0	1.25
Diagnostic certainty (type and location of lesions described)	5	5	5	5	5	5	5	5	5
Description of surgical procedure given	5	3	0	5	5	5	5	5	4.125
Description of postoperative rehabilitation	10	0	0	0	0	10	10	10	5
Part B. Scores may be given for each option in each of the 3 sections if applicable
Outcome criteria	2	5	5	5	7	5	5	7	5.125
Procedure for assessing outcomes	0	0	6	6	10	6	0	7	4.375
Description of subject selection process	8	5	5	5	8	0	5	5	5.125
Total score	40	48	46	41	50	53	55	54	48.375

NOTE. Shown is the Modified Coleman Methodology Score for the 8 human clinical studies.

Study Population Size

The papers totaled 397 patients with a mean of 49.6 patients each. The number of patients ranged from 8 to 111 patients. Means score for this component was 5.

Follow-Up

This component had a mean methodology score of 5. Five studies had a mean follow-up time greater than 24 months, compared with one with a mean between 12 and 24 months and 1 with less than 12 months. The mean score for this component of the score methodology was 3.375.

Surgical Sites

All studies only included patients who underwent 1 surgery for each reported outcome. All articles looked at only knees except Cinats et al.,¹⁸ which included both shoulder (n = 3) and knee (n = 17) OCA transplants. The mean score for this component was 10.

Study Type and Levels of Evidence

Three articles were retrospective reviews, 4 articles were case series studies, and 1 was a prospective study. There were no randomized control studies included. The mean score for this component was 1.25. Overall, the level of evidence for the included studies is 4 articles with Level of Evidence III and 4 articles with Level of Evidence IV.

Diagnostic Certainty

All studies included information regarding the type and location of the OCA transplant. The mean score for this component was 5.

Description of Study Reported

Six studies included extensive detailed surgical detail, whereas one mentioned but did not elaborate on the surgical details. One study did not give any details about the surgery. The mean score for this component was a 4.125.

Ex Vivo

In total, 19 ex vivo studies were included in this study. Ex vivo studies were evaluated on the basis of the important components of the storage process for OCAs including tissue source and type, storage procedure including temperature, media changes, gas environment, storage solution including type of solution and addictive used, cell viability methodology, and outcomes methodology. A heat map of these major criteria can be found in Figure 2. Components of each section are included as nonbolded text.

Fig 2.

Heat map of osteochondral allograft storage solutions. Shown are commonly used storage solutions and additives were used for each ex vivo study. Green indicates that the criteria was reported/used. White indicates criteria that it was not reported/used.

All the studies included tissue source and tissue type used, with 9 (47.37%) from joint arthroplasty and 12 (63.16%) from donor cadavers. Two studies, Sun et al.¹⁹ and Hevesi et al.,²⁰ used both cadavers and surgical tissue in their study. Predominantly, whole or cut osteochondral tissue was used (57.89%) followed by cartilage biopsies/plugs (31.58%). In 1 study, Robertson et al.²¹ used an osteochondral core. Only 47.37% of the studies reported the size of the tissues. Only 4 (21.05%) reported gas environment information. A heat map of these variables can be found in Figure 3.

Fig 3.

Heat map of osteochondral allograft storage variables. Shown is osteochondral specimen information and which storage condition were used for each ex vivo study. Green indicates that the criteria was reported/used. White indicates criteria that it was not reported/used.

Cell Viability

All except one study included information about cell viability. Commonly used cell viability methods include flow cytometry, fluorescence stain on cartilage, cell fluorescence of cell isolates, water-soluble tetrazolium assay, and MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) assay. Flow cytometry uses laser-based methods to detect and analyze the chemical and physical character of cells to determine cell viability. Fluorescence stain on cartilage and cell fluorescence of cell isolates use the intensity of fluorescence of biomarkers and cell membrane proteins to determine cell viability with the only difference between the 2 being if it is performed on tissue cartilage samples or isolated cells. Water-soluble tetrazolium and MTT are 2 very similar assays that use different tetrazolium salts to determine metabolic activity of cells. The most used methods were cell fluorescence on cartilage (68.42%) and cell fluorescence of cell isolates (15.79%). The most used outcome methodology was tissue histologic analysis with 7 (36.84%) studies.

Storage Media

Two studies, Csönge et al.²² and de Sousa et al.,²³ included all storage information (solution formulation, volume, container, and type of media). However, all but one included storage media solution type that was used. Storage media solutions used are included in Table 3. The most used media were Eagles Minimum Essential Medium, dimethyl sulfoxide, serum-free media, and RPMI-1640. These media were used in 3 studies each (Table 3). Two studies used Missouri Osteochondral Allograft Preservation System (MOPS) and phosphate-buffered saline each (Table 3). Thirteen (68.42%) of the studies also included additives to the storage media solutions, the most common of which were antibiotic/antimycotics (10) and L-glutamine (5).

Table 3.

Commonly Used Storage Solutions

Solution	Total Studies
Eagles Minimum Essential Medium (EMEM)	3
Dimethyl sulfoxide (DMSO)	3
Serum-free media (SFM)	3
RPMI-1640	3
Missouri Osteochondral Allograft Preservation System (MOPS)	2
Phosphate-buffered saline	2
Dulbecco’s Modified Eagle Medium (DMEM)	1
Dulbecco's Modified Eagle Medium: Nutrient Mixture F12 (DMEM F12)	1
Fetal bovine serum	1
Iscove’s Modified Dulbecco’s medium (IMDM)	1
Lactated Ringer’s solution	1
Minimal Essential Medium Eagles (MEME)	1
N-acetylcysteine	1
X-Vivo 10	1
Chondrocyte growth medium (CGM)	0
Euro-Collins (EC) solution	0
INCELL media	0
NCTC medium	0
University of Wisconsin (UW) solution	0

NOTE. Shown are commonly used storage media for osteochondral allografts and the number of identified studies that used each.

In terms of storage media, Calvo et al.²⁴ found N-acetylcysteine enriched medium to provided improved cell viability compared to basal medium. In 2 studies, Bae et al.²⁵ and Han et al.²⁶ found the RPMI storage with (−) epigallocatechin-3-O-gellate additive to have significantly maintained OCA cell viability. Ball et al.²⁷ showed that use of Dulbecco’s Modified Eagle’s Medium (DMEM)/Ham’s Nutrient Mixture F12 with supplemental L-glutamine, ascorbic acid, streptomycin, penicillin, and amphotericin enhanced OCA viability and metabolic activity compared to OCA storage in lactated Ringer’s solution.

Storage Temperature

All but one study reported temperature information, with most stored at 4°C or 1 to 10°C (78.95%). In total, 31.58% and 21.05% were stored at 37°C and room temperature, respectively. Of the 19 studies, 3 specifically compared storage of solution at different temperatures, 4 specifically compared different storage media, and 2 compared both storage media and temperature. de Sousa et al.²³ and Lin et al.²⁸ found significant improvement in OCA cell viability storing the solution at 37°C compared to 4°C. Denbeigh et al.²⁹ showed solutions stored at 37°C had increased OCA viability and metabolism as well as maintained collagen expression compared with 22°C. Use of MOPS storage media at 25°C was shown to have greater OCA viability and lower inflammatory mediators than those stored using tissue bank protocols at 4°C.^30,31

Discussion

Key findings in this systematic review include OCA storage at 4°C or 1 to 10°C remains the most common storage temperature with the most evidence-based research supporting its efficacy, with 78.95% of included studies investigating this temperature. However, investigation at 37°C and room temperature, particularly those stored with proprietary processing protocols over the past several years, have shown promising and even superior results at improved maintenance of viable chondrocyte density compared to 4°C. There remains much variability in storage media protocols, with 19 different storage media investigated, 68.42% containing various additives, highlighting a lack of clear consensus on media additives. Moreover, newer research in orthobiologic augmentation of graft preparation with bone marrow aspirate concentrate (BMAC) and platelet rich plasma has shown promising results, although further investigation is needed to address the paucity of research human-subject studies in this area and resolve conflicting evidence.

Historically, OCA storage methodology has evolved over time to meet several discrete goals using the best-available evidence. Patient safety has remained first and foremost the primary objective, ensuring patients obtain grafts stored via best practices to avoid complications. Another integral goal of research is increasing the length of storage of grafts while maintaining graft efficacy to maximize availability of the procedure for patients and increase the limited resource of grafts. The key to improved graft storage time is the maintenance of chondrocyte viability, which is a crucial metric in OCA storage literature. Greater than 70% chondrocyte viability at time of OCA implantation is a commonly accepted threshold.³⁰ Moreover, in the investigation of a variety of graft preparation and storage protocols, cost-effectiveness has remained an important consideration to maximize availability of the treatment option without being financially prohibitive.

Processing: Donor Selection

In the United States, the Food and Drug Administration oversees the procurement and processing of OCAs.³² The Food and Drug Administration issues Good Tissue Practices, as guidelines for facilities handling human tissues to use when appropriate to regulate and minimize the transmission of communicable diseases.³³ Ideal donors are aged 15 to 40 years of age by convention, with harvest happening within 24 hours of donor death with en bloc aseptic resection.^34,35 Interestingly, no clinical study has reported an ideal donor age thus far using patient outcomes. Nuelle et al.¹⁶ in fact found no significant difference in donor age between patients with a successful verse unsuccessful OCA outcome, though the age range of donors was relatively narrow at 15-32 years. Similarly, Rauck et al.¹³ did not find a difference in donor age between those with grafts that had chondral delamination from the cancellous bone surface, one of the documented modes of OCA failure, versus those that didn’t. Logistic and scalability problems arise as OCAs are typically obtained from young deceased donors, with limited availability.

Donors are screened for high-risk activity, and after harvest, grafts undergo mandatory serologic and microbiologic testing to reduce rates of transmissible blood borne viruses such as hepatitis C, hepatitis B, and HIV.^32,36,37 Grafts are in turn measured for size by tissue banks. Although most of treated cartilage defects are on the medial femoral condyle, only 25% of available grafts are from medial femoral hemicondyles.³⁸ Although graft-recipient matching has been favored to optimize articular congruity, in clinical study there has been no significant difference in clinical outcomes for orthopneic grafts taken from an alternate condyle than its recipient.³⁹ Thus, condyle-specific matching may not be necessary. Anteroposterior matches have also been targeted to improve articular incongruity and reduce mismatch in radius of curvature, potentially decreasing susceptibility of graft failure.⁴⁰ However, a study of 69 patients found magnitude of graft-recipient anteroposterior mismatch to not be associated with graft failure or outcomes.⁴⁰

Storage Duration

Length of graft storage before transplantation is a key factor in OCA use, with grafts being released after a 14-day quarantine to allow for infectious disease screening.³³ There is a litany of ex vivo studies showing that graft chondrocyte viability decreases over time.⁴¹ In analyzing 60 osteochondral grafts, Williams et al.⁴² discovered significantly decreased levels of both chondrocyte viability and proteoglycan synthesis at 28 days postharvest, even though the cartilage matrix remained maintained. Moreover, Nuelle et al.¹⁶ in a study of 75 patients discovered clinically that graft storage duration did in fact have a significant effect on successful OCA patient outcomes, with patients with grafts stored more than 28 days before transplantation being 2.6 times more likely to have an unsuccessful outcome. Merkely et al.¹⁷ in 2020 found patients who received OCA grafts stored for 25 to 27 days had significantly lower graft survivorship at 5 years follow-up compared with those with grafts stored 19 to 24 days (70.3% survivorship vs 93.1%, respectively). However, at shorter storage time points, in a 2017 study, Schmidt et al.¹⁵ found no significant difference in 5-year graft survivorship between those used an average of 6.3 versus 20.0 days. As storage time increases, an upregulation of proapoptotic gene expression occurs leading to chondrocyte death.²¹

The effect of storage length on graft failure outcomes in human clinical studies is detailed in Figure 4.15, 16, 17 The effect of storage length on cell viability in ex vivo studies is detailed in Figure 5.^21,41, 42, 43, 44

Fig 4.

The effect of storage length on graft failure outcomes in human clinical studies. Shown are storage length outcomes for three human clinical studies that investigated storage length effect on OCA failures. Lines between points indicate the day ranges of experimental groups used by the studies.

Fig 5.

The effect of storage length on cell viability in ex vivo studies. Shown are storage length outcomes for ex vivo studies that investigated storage length effect on either cell viability or cell molecular analysis. Lines between points indicate the day ranges of experimental groups used by the studies.

Storage Temperature

Storage temperature plays a key role in maintaining viable chondrocyte density. Although frozen grafts allow the benefit of allowing for transport to further locations prior to thawing, crystallization that occurs during cooling and warming phases can result in cell death, and they have repeatedly shown lower chondrocyte viability.^22,45 Other protocols have explored cold storage at 4°C, which is the most common temperature of storage in this systematic review, though research indicates significant time-dependent loss of chondrocyte viability, with decreased expression of collagen and increased expression of “early response” genes that mediate stress.^24,43,46 However, there is a mounting body of evidence of improved graft characteristics when stored at warmer temperatures.^30,31 de Sousa et al.²³ found grafts stored at 4°C to have lower cellular mortality at 14 days when compared with those at 37°C. When compared with grafts stored at 22°C, those stored at 37°C may again have improved viable chondrocyte density.²⁵

Nutrient Media

Media choice has proven to be an important factor influencing graft cell viability, without a clear consensus on preferred protocol (Fig 3). This systematic review identified various media additives used in 68.42% of human ex vivo studies, highlighting this lack of consensus. In 2004, Ball et al.²⁷ reported culture media (DMEM, a broadly used culture medium enhanced with amino acids and vitamins) to provide a better environment compared with lactated Ringer’s at 2 weeks postharvest.³⁴ Enriched storage media with 1 mM of N-acetylcysteine has been associated with improved cell viability.²⁶ In addition to being associated with preserved chondrocyte viability, OCAs with (−) epigallocatechin-3-O-gellate have also been found to have well-preserved cartilaginous structures with delayed denaturation of the extracellular matrix.^27,28 Cultures with human albumin have also been associated with improved chondrocyte survival.²³ Storage in fetal bovine serum has been associated with improved chondrocyte viability and density compared with serum-free media, though with it a possibility of infectious disease transmission risks and immunogenic factors, raising the importance of graft lavage.⁴⁴

Lavage

Lavaging OCAs removes donor antigentic marrow that can illicit an immune response.⁴⁷ OCA marrow cleansing progresses from the base toward the cartilage, and has been found to increase with lavage duration and flow intensity.¹⁹ However, pulsed lavage has also been found to have limited effectiveness at removing marrow elements.⁴⁸ Recent research has found combination saline and high-pressure CO₂ lavage to more effectively remove marrow elements from OCAs than saline alone.⁴⁹ In an effort to minimize patients’ postoperative pain after OCA surgery, investigators have explored washing grafts in analgesic cocktails. In an ex vivo study, Baumann et al.⁵⁰ have reported that washing the graft with a combination of morphine, ropivacaine, epinephrine, and ketorolac had no significant derogatory effect on chondrocyte cell viability relative to controls, yet did result in a significant decrease in expression of degradative and inflammatory mediators, PGE2, MCP-1, MMP-7, and MMP-8.

Orthobiologic Preparation

After lavage, some surgeons are adding orthobiologics before transplantation to improve healing and graft incorporation.⁵¹ Successful clinical outcomes have been obtained using bone marrow aspirate concentrate as part of graft preparation with application to the graft’s subchondral bone.⁵² Oladeji et al.⁵³ have found the use of BMAC to be associated with significantly higher graft integration at 6 weeks, 3 months, and 6 months postoperatively. However, there is still a paucity of literature on BMAC application to OCAs in human studies, with some conflicting evidence relative to its effectiveness.^54,55 In animal models, platelet-rich plasma addition to OCA preparation has begun to be explored, but there is a lack of consensus and studies in humans.^56,57

MOPS Protocols

MOPS has been an intriguing and novel protocol, storing grafts in proprietary media at room temperature, with maintained cell viability at longer storage times than standard tissue bank protocols.¹⁴ The MOPS protocol uses proprietary media which includes a combination of DMEM, glucose, dexamethasone, and other additives.⁵⁸ In a sample of 50 patients, grafts stored with MOPS protocol at an average storage of 44.2 days had no significant difference in viable chondrocyte density (VCD) compared with normal healthy cartilage, and 100% of grafts were above a 70% minimum chondrocyte density at time of transplant, compared with just 27% of grafts stored with standard protocols meeting this threshold. Moreover, the VCD was significantly greater in the MOPS group. In a follow-up study, grafts stored with MOPS protocol at 25°C maintained their day 0 VCD through 56 days of storage, and were significantly higher than standard tissue bank protocols at 4°C by day 28.³⁰ However, histologic and biomechanical properties did not differ over time for either group. Grafts stored using MOPS protocol have also been found to release lower levels of inflammatory mediators and degradative enzymes, one proposed mechanism contributing to graft failure.³¹ A 2023 study using MOPS protocol on whole-surface shell OCAs for patellofemoral lesions, in conjunction with BMAC augmentation of grafts prior to implantation, showed significantly greater graft survival at mean follow-up of 44.8 months and significantly fewer reoperations when compared with grafts cold stored with standard preservation protocols.⁵⁹

Limitations

This systematic review has some limitations. The primary limitation is the high heterogeneity of ex vivo methodologies used, particularly in storage media protocol. Moreover, there was a lack of randomized controlled trials identified in human clinical studies that investigated storage methodology variables. A further limitation is the possibility of this review’s search methodology not capturing a relevant study.

Conclusions

OCA storage at 4°C remains the most common temperature with the most evidence-based research. However, investigation of OCAs at 37°C and room temperature, particularly those stored with proprietary protocol such as the MOPS, have shown promising results at improved maintenance of viable chondrocyte density. Variability in storage media remains without clear consensus.

Disclosures

The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: N.S. reports the publication fee was provided by Northwell Health. All other authors (M.J.M., K.P.) declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.