Abstract
Meniscal allograft transplantation may be a better alternative for the treatment of irreparable meniscal injury compared to other forms of treatment. However, it remains to be seen whether the use fresh frozen allograft is better than cryopreserved allograft in treating this type of injury. We hypothesized that cryopreserved meniscal allograft would work better in maintaining the original biomechanical properties compared to fresh frozen ones, due to the lower amount of damage it incurs during the storage process. We examined young and healthy human menisci obtained from orthopedic oncology patients who underwent resection surgeries around the knee. The menisci obtained were preserved via cryopreservation and deep-freezing process. Traction tests were carried out on the menisci after 6 weeks of preservation. Twelve pairs (N = 24) of menisci were divided equally into two groups, cryopreservation and deep frozen. There were six males and six female menisci donors for this study. The age range was between 15 and 35 years old (24.9 ± 8.6 years). Cryopreserved specimens had a higher ultimate tensile strength (UTS; 8.2 ± 1.3 Mpa vs. 13.3 ± 1.7 Mpa: p < 0.05) and elastic modulus (61.7 ± 27.6 Mpa vs. 87.0 ± 44.10 Mpa: p < 0.05) compared to the fresh frozen specimens. There was a significant difference in UTS (p < 0.05) between the two groups but no significant difference in their elastic modulus (p > 0.05). The elastic modulus of the preserved meniscus was similar to fresh normal menisci taken from other studies (60–120 Mpa; cryopreserved (87.0 ± 44.1) and fresh frozen (61.7 ± 27.5)). Cryopreserved menisci had a higher elastic modulus and point of rupture (UTS) compared to fresh frozen menisci. Cryopreservation proved to be a significantly better method of preservation, among the two methods of preservation in this study.
Introduction
Meniscal replacement is becoming the treatment of choice among sports medicine surgeons, to treat meniscal deficient knee. The importance of meniscus in the protection of articular cartilage of the knee was first described by Fairbanks, 1 followed by Baratz. 2 Arnoczky et al. 3 was the first to replace a cryopreserved meniscal allograft in dogs and Milachowski et al. 4 was the first to replace meniscus in humans. Meniscal allograft transplantation may be a better alternative compared to other forms of treatment for irreparable meniscal injury. 2,5,6 It has been shown that type of preservation method used does affect the quality of the meniscus. 6 –8
The indication for a successful meniscus transplantation is protection of the articular cartilage, pain relief, re-establishing normal kinematics (transmit force, distribution load, and stabilize the joint), and causing no harm to the joint either directly or indirectly at a later time. The most important feature that is generally required in replacement materials is the ability to resist biomechanical stresses. 5 –7 A good replacement material must also provide biomechanical properties comparative to that of native meniscus. 9 –14
Although there is an increase in the use of meniscus worldwide, there is still a lack clinical and scientific focus on aspects such as finite element models and biomechanical analysis of meniscal allografts. Allogeneic meniscus has been said to provide good biomechanical properties, but the best method of its preservation remains to be determined. 5,6,8,11
We hypothesized that cryopreserved meniscal allograft would maintain the original biomechanical properties better compared to fresh frozen allografts, due to the lower amount of damage it incurs during the storage process. This hypothesis, however, had not been investigated before the inception of this study. There was no clinical report of direct comparison between deep fresh freezing and cryopreservation of young, healthy human menisci in a single biomechanical study. Considering that these are the most common methods of preservation, we conducted this study to determine which of these two methods would preserve the biomechanical properties of the menisci better. Both types of preservations are commonly used and have proven to give good clinical outcome. 5,7,11
Material and methods
This study was conducted in University Malaya Medical Centre (UMMC), Kuala Lumpur, Malaysia, from June 1, 2011 till September 30, 2012 (16 months). It was conducted in two phases. Phase 1 consisted of procurement and storage followed by biomechanical testing in phase 2. Ethical approval was obtained from the Hospital research committee prior to conducting this research (MEC ref no: 872.15). Informed consent was obtained from all individual participants included in this study.
The grafts procured for this study were from orthopedic oncology patients of UMMC, who were undergoing resection and endoprosthesis replacement around the knee. The exclusion criteria for this study were as follows: age of more than 45 years old, 15 knee degenerative changes, 2 tumor with intra-articular extension into the knee, 16 meniscal deficient knee, and a history of knee septic arthritis or infections around the knee. The surgical procedure, procurement of their meniscus, and the experiment were explained to the study subjects with a detail patient information sheet, after which written consent was obtained from each of the subjects.
On the day of the surgery, after wide resection of the tumor, the meniscus was isolated and procurement was done under aseptic technique. 17 All harvested menisci were firstly washed with saline to remove all traces of blood and its constituents (Figure 1). This step is undertaken to reduce its immunogenicity. 18,19 All unnecessary fatty tissue attachments were also removed from the grafts. This procedure was performed under magnification for more accurate cleansing followed by measurements. All unwanted tissues were then sent for histopathology, culture, and sensitivity analyses. Any graft found to have traces of tumor material or bacterial colonization was discarded. All the above procedures were performed in the same operation theater as the resection surgery. All grafts used in this study were not irradiated as gamma irradiation is shown to reduce the allograft strength. 20
The harvested meniscus.
All prepared menisci were then measured with digital caliper and weighed with an electronic balance device. The digital caliper and electronic balance had a minimal measurement of 0.01 mm and 0.01 g, respectively. Strict criteria were used when measuring all menisci. Only one investigator took all the measurements and a standardized method was used to measure where the caliper used had to create a mild indentation before any measurement was recorded. Measurements were taken in triplicates. The mean value was finalized as the final measurement. The measurements taken were length, peripheral length width, and thickness. The latter two were measured at three separate areas, namely, anterior one-third, middle one-third, and posterior one-third. Grafts were then randomly stored using either cryopreservation or deep-freezing methods at −80 to 90°C temperatures. Randomization was carried out via drawing lots, and it was done before the procurement. All menisci were stored for 6 weeks 8 before they were thawed and tested.
There were two types of freezing methods used in this study, namely, cryopreservation at temperatures as low as −180 to 190°C and deep freezing at temperatures of −80 to 90°C. The specimens for cryopreservation were pretreated with glycerol. In this study, 20% glycerol in cryo-tanks was used. Cryopreservation and fresh freezing preservations were performed per standards set by the international tissue allograft preservations. 5,7,14 The specimens for both forms of preservations were thawed after 6 weeks of storage. Fresh frozen allografts were thawed with saline to eliminate the crystal water content. 5 Cryopreserved allografts were thawed with ringer’s lactate solution to preserve cellular viability. Both specimens were thawed at room temperatures for 4–6 h before testing. 5 The specimens were not thawed any more than five times to prevent any cellular damages that might compromise the biomechanics of allograft. 21
After the thawing process, all menisci allografts were measured and prepared for testing. 15 The outer layer of the menisci was cut using a blade, in a vertical and stepwise approach, considering the greasy and slippery nature of the menisci. This was used to test the biomechanics of the outer part of the meniscus where the tensile forces occur.
In this study, we only tested stress versus strain due to the limitation of sample size. All tests were carried out using the Instron machine (microtester 5848, Instron; Figure 2). The end point of the test was a tear at the center of meniscus (Figure 3). During the preliminary tests, the menisci tend to slip from each side of the grip, thus affecting the results of the test. Therefore, suture, resin, or fiberglass were tested individually and in combination, on the menisci, to determine the best method to prevent slipping of the graft. The combinations of “suture–resin” and “fiberglass–suture” proved to be the best combinations to counter the slipping. In this study, the combination of “suture–resin” was used. After preparation, the medial part of the meniscus that was not involved in the mechanical testing was weighed to measure the water content, to compare between the two forms of preservations. 8 All menisci were weighed both before and after being dried. The caliper gauge used was Digimatic Caliper (Mitutoyo Corporation, Tokyo, Japan) with a measuring range of 0–150 mm and minimum indication of 0.01 mm.
Graft is secured on both ends of the Instron machine and pulled.
Rupture of the midportion of the graft is shown.
Stress versus strain graphs were then formulated after traction testing. Medial and lateral graphs for both preservation methods were also plotted. Ultimate tensile strength (UTS) and elastic modulus, E, were then identified. ANOVA statistical test, followed by post hoc test with Bonferoni correction were applied for both UTS and elastic modulus values.
Results
There was a total of 12 pairs of menisci tested in this study. These menisci were divided into two groups, namely, cryopreservation and fresh frozen, consisting of six pairs in each group. All but one pair of menisci were taken from patients with osteosarcoma. The single pair of menisci was obtained from a patient with giant cell tumor of the bone. The age of the study subjects ranged between 15 and 35 years, with a mean age of 24.9 ± 8.6 years. There were equal numbers of male and female (N = 6 each) in this study.
Table 1 shows that the sum of total water content increased the size of the menisci in both medial and lateral menisci for both forms of preservations.
Mean measurements pre- and post-preservation.
SD: standard deviation.
Table 2 shows the value obtained from measuring the dry weight for both medial and lateral menisci in both preservations and calculating the mean for each preservation method. Descriptive statistical analysis shows that the sum of total for water content for cryopreservation was 60.4% ± 0.96%, compared to 63.8% ± 1.63% (post hoc power analysis = 3.7%) for fresh freezing method.
Dry weight (g)—Comparison before and after preservations for water content tests.a
a p Value of mean different for both preservation was less than 0.01.
From these tests, it was found that the medial menisci from the cryopreservation group had a higher maximum stress level compared to other groups (Table 3). The mean UTS for cryopreserved (both medial and lateral) versus fresh freezing methods was calculated (13.34 ± 1.74 vs. 8.18 ± 1.31; p < 0.05, post hoc power analysis = 6%). Cryopreserved menisci also showed a higher elastic modulus when compared to fresh frozen samples (Table 4). The mean for cryopreserved (both medial and lateral) versus fresh freezing method was also calculated (86.99 ± 44.09 vs. 61.67 ± 27.55; p > 0.05, post hoc power analysis = 28.6%). There were no failures of the meniscus–suture–resin system.
Mean and comparison of UTS with different preservation in both medial and lateral meniscus using post hoc test.
SD: standard deviation; UTS: ultimate tensile strength.
Mean and comparison of Young’s modulus with different preservation in both medial and lateral meniscus using post hoc test.
SD: standard deviation.
Discussion
Biomechanics
The focus of this investigation is on the mechanical testing of the meniscal materials after preservation with the two commonest methods. This study was to determine whether cryopreservation is a better option for preservation compared to fresh freezing for menisci biomechanically. In our study, we used elastic modulus and UTS to determine the efficacy of the method of preservation. The elastic modulus was determined using stress versus strain graphs. The mean for both medial and lateral menisci preserved in cryopreservation and fresh freezing was calculated (as shown in Table 4). The elastic modulus for cryopreservation was significantly better than the elastic modulus for fresh frozen samples. Predrag 15 studied the elastic modulus for their fresh freezing meniscus and found that it varied from 20.3 MPa to 129.1 MPa. However, their study did not analyze cryopreserved meniscus.
Comparison was done between the data of this study and the published data on fresh normal meniscus. Published literature 22,23 described the elastic modulus for the normal, fresh, young menisci as 60–125 Mpa. The cryopreservation group was within the range for the normal young menisci (86.99 ± 44.10) but elastic modulus for fresh freezing showed a much lower (61.67 ± 27.54). These findings favor cryopreservation. This is probably because cell membrane integrity and donor chondrocyte viability is well preserved in the presence of glycerol in cryopreservation. Glycerol is a cryoprotectant that prevents the formation of ice inside the cell. Cryoprotectants facilitate cell preservation by minimizing the cell water content, supporting vitrification occurrence, thus protecting the cell’s constitutive macromolecules. About 20–30% glycerol is usually sufficient to cryoprotect most crystallized proteins. 5 Freezing on the other hand may affect the donors’ cells, thus altering their meniscus biomechanics.
Another factor analyzed was UTS. This is the ability of collagen tissue to absorb energy until it ruptures, and it is also known as the toughness of the tissue. It was determined by measuring the area under the stress versus strain curve. A study of that area showed that it takes a certain amount of energy to reach plastic deformation and it takes a relatively large amount of energy to reach failure. This provides definite safety against rupture. Important material parameters can be extracted from the stress–strain relationship. The outer part of the meniscus was used to test the biomechanics in this study, as this was the site where most of the tensile forces occurred. The point of failure at maximum tension of the menisci is called UTS. This point was plotted inside stress versus strain graphs and results were taken for comparison. The mean for UTS for both medial and lateral menisci preserved in cryopreserved and fresh freezing was calculated. The values were 13.34 ± 1.74 Mpa and 8.18 ± 1.31 Mpa, respectively, and the p value was less than 0.05. Higher values were recorded by Tissakht in his study for fresh freezing preservation, with a mean of 19.81 ± 4.40. 24 These higher values might be due to variations in specimen size, cutting method used, and area of the tests. Lower values for post hoc power test were most likely because of smaller sample size.
Measurements, weight, and dry weight
The cryoprotectant usually maintains the size of the specimens. Comparisons were made on the size and weight of specimens, pre- and post-preservation. The measurements showed that the menisci were slightly larger in both forms of preservation. This might be due to freezing and thawing processes. The freeze–thaw process contributes to double stress in the form of thermal and hyperosmotic stress, which act simultaneously during cooling, on the graft cells. 25 In the process of cryo-freezing, the water surrounding the cell freezes first before the cell contents. This is because the cytoplasm is more concentrated than the growth medium and per thermodynamics, the component with largest volume will nucleate first. This will increase the osmotic pressure and extracellular solute becomes concentrated in the remaining liquid extracellular water. Subsequently, osmosis will dehydrate the cells as water diffuses from the cytoplasm into more concentrated external solution.
If the outflow of water is faster than the heat flow, then progressively, as the temperature decreases, the water contained in the medium will freeze. That is why it is better to have a gradual, controlled cryo-container temperature so that the outflow of water is more, thus the tissue remains viable. There was no gradual control of temperature in cryopreservation tank in this study. 14,25 Furthermore, in the process of thawing in the room temperature, the process of freezing is reversed. This restores the original size of the meniscus. However, with the addition of ringers lactate, the tissue sometimes becomes larger than its original size. The fresh frozen technique utilizes a freezer machine to deep freeze the graft (−80°C). This causes all the water (both intra and extra cellular) to crystalize and damage the cells and tissue. The thawing process at room temperature with normal saline also results in increase in size of the meniscus.
This study showed that pre- and post-freezing and cryopreservation caused an increase in size of the menisci. In terms of water content for post preservation, results obtained showed that cryopreservation had a slightly lower amount of water compared to freezing technique. Therefore, the increase in size post-preservation in cryopreservation method was mainly due to the water content. Statistically, there was no significant difference between the two preservation methods in term of measurements of size, but there was a significant difference between water contents for both preservations.
Limitations of this study include a small sample size and the variation in age range for young menisci of less than 35 years old. The normal, unpreserved meniscus was not tested due to the limited availability of the sample meniscus, thus previously published study results were used for comparison. We studied the size and water content of post-preservation menisci specifically and not post-transplantation meniscus. Post transplantation, the size of menisci might decrease. The average amount of shrinkage might be up to 7%. 26,27 We also did not look at the cell distribution. Deep frozen allografts would have the least amount of cell density compared to other viable allografts and polyurethane scaffolds. 27,28
Conclusion
Cryopreserved menisci showed a higher elastic modulus and point of rupture UTS. There was significant difference between the two methods of preservations in this study and it favors cryopreservation. Therefore, we recommend the use of cryopreserved meniscus as a method of preservation for meniscus as it retains the meniscus biomechanical properties.
Footnotes
Declaration of conflicting interests
The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
Funding
The author(s) received no financial support for the research, authorship, and/or publication of this article.