CircPVT1 promotes migration and invasion by regulating miR-490-5p/HAVCR2 axis in osteosarcoma cells.

Circular RNAs (circRNAs) play an important role in the progression of osteosarcoma. However, the precise function of circPVT1 in osteosarcoma remains elusive. This study aims to explore the molecular mechanism underlying the involvement of circPVT1 in osteosarcoma cells. We quantified circPVT1 expression using qRT-PCR in both control and osteosarcoma cell lines. To investigate the roles of circPVT1, miR-490-5p and HAVCR2 in vitro, we separately conducted overexpression and inhibition experiments for circPVT1, miR-490-5p and HAVCR2 in HOS and U2OS cells. Cell migration was assessed through wound healing and transwell migration assays, and invasion was measured via the Matrigel invasion assay. To elucidate the regulatory mechanism of circPVT1 in osteosarcoma, a comprehensive approach was employed, including fluorescence in situ hybridization, qRT-PCR, Western blot, bioinformatics, dual-luciferase reporter assay and rescue assay. CircPVT1 expression in osteosarcoma cell lines surpassed that in control cells. The depletion of circPVT1 resulted in a notable reduction in the in vitro migration and invasion of osteosarcoma cells. Mechanism experiments revealed that circPVT1 functioned as a miR-490-5p sequester, and directly targeted HAVCR2. Overexpression of miR-490-5p led to a significant attenuation of migration and invasion of osteosarcoma cells, whereas HAVCR2 overexpression had the opposite effect, promoting these abilities. Additionally, circPVT1 upregulated HAVCR2 expression via sequestering miR-490-5p, thereby orchestrating the migration and invasion in osteosarcoma cells. CircPVT1 orchestrates osteosarcoma migration and invasion by regulating the miR-490-5p/HAVCR2 axis, underscoring its potential as a promising therapeutic target for osteosarcoma.

Compressive mechanical properties of vitrified porcine menisci are superior to frozen and similar to fresh porcine menisci.

The common practice of freezing meniscal allograft tissue is limited due to the formation of damaging ice crystals. Vitrification, which eliminates the formation of damaging ice crystals, may allow the mechanical properties of meniscal allograft tissue to be maintained during storage and long-term preservation. The primary objective of this study was to investigate the differences between fresh, frozen, and vitrified porcine lateral menisci examining compressive mechanical properties in the axial direction. Unconfined compressive stress-relaxation testing was conducted to quantify the mechanical properties of fresh, frozen and vitrified porcine lateral menisci. The compressive mechanical properties investigated were peak and equilibrium stress, secant, instantaneous and equilibrium modulus, percent stress-relaxation, and relaxation time constants from three-term Prony series. Frozen menisci exhibited inferior compressive mechanical properties in comparison with fresh menisci (significant differences in peak and equilibrium stress, and secant, instantaneous and equilibrium modulus) and vitrified menisci (significant differences in peak stress, and secant and instantaneous modulus). Interestingly, fresh and vitrified menisci exhibited comparable compressive mechanical properties (stress, modulus and relaxation parameters). These findings are significant because (1) vitrification was successful in maintaining mechanical properties at values similar to fresh menisci, (2) compressive mechanical properties of fresh menisci were characterized providing a baseline for future research, and (3) freezing affected mechanical properties confirming that freezing should be used with caution in future investigations of meniscal mechanical properties. Vitrification was superior to freezing for preserving compressive mechanical properties of menisci which is an important advance for vitrification as a preservation option for meniscal allograft transplantation.

Effect of storage media on chondrocyte viability during cold storage of osteochondral dowels.

Osteochondral allograft transplantation is a successfully proven method to repair articular cartilage defects and prevent the degenerative effects of osteoarthritis. The number of osteochondral transplantations that can be performed each year is limited by availability of donor cartilage tissue and storage time constraints. Osteochondral transplantation success has been linked to high chondrocyte viability of the donor cartilage tissue at the time of implantation. Determining optimal storage conditions for donor cartilage is essential for tissue banks to safely provide quality cartilage tissue. In this study, we compared three tissue/cell media (DMEM/F12, RPMI-1640 and X-VIVO 10) for their ability to maintain chondrocyte viability during hypothermic storage for 28 days. Porcine osteochondral dowels were stored in each media for 28 days and cell viability was assessed every 7 days. Over the 28 day storage period, the chondrocyte viability of dowels stored in DMEM/F12, RPMI-1640, and X-VIVO 10 media all declined in a similar fashion. Our results show that all three media were equivalent in their ability to maintain cell viability of the cartilage tissue and provides rationale for the use of lower cost cell media (DMEM/F12 and RPMI-1640) for hypothermic storage of articular cartilage tissue.

The effect of sucrose supplementation on chondrocyte viability in porcine articular cartilage following vitrification.

Vitrification can extend the banking life of articular cartilage (AC) and improve osteochondral transplantation success. Current vitrification protocols require optimization to enable them to be implemented in clinical practice. Sucrose as a non-permeating cryoprotective agent (CPA) and clinical grade chondroitin sulfate (CS) and ascorbic acid (AA) as antioxidants were investigated for their ability to improve a current vitrification protocol for AC. The aim of this study was to assess the impact of sucrose and CS/AA supplementation on post-warming chondrocyte viability in vitrified AC. Porcine osteochondral dowels were randomly vitrified and warmed with one established protocol (Protocol 1) and seven modified protocols (Protocols 2-8) followed by chondrocyte viability assessment. Sucrose supplementation in both vitrification and warming media (Protocol 4) resulted in significantly higher (p = 0.018) post-warming chondrocyte viability compared to the protocol without sucrose (Protocol 1). There was no significant difference (p = 0.298) in terms of post-warming chondrocyte viability between sucrose-supplemented DMEM + CS solution (Protocol 4) and Unisol-CV (UCV) + CS (Protocol 6) solution. Clinical grade CS and AA contributed to similar post-warming chondrocyte viability to previous studies using research grade CS and AA, indicating their suitability for clinical use. The addition of an initial step (step 0) to reduce the initial concentration of CPAs to minimize osmotic effects did not enhance chondrocyte viability in the superficial layer of AC. In conclusion, sucrose-supplemented DMEM + clinical grade CS (Protocol 4) could be an ideal protocol to be investigated for future use in clinical applications involving vitrified AC.

Evaluation of the permeation kinetics of formamide in porcine articular cartilage.

Cryopreservation of articular cartilage will increase tissue availability for osteochondral allografting and improve clinical outcomes. However, successful cryopreservation of articular cartilage requires the precise determination of cryoprotectant permeation kinetics to develop effective vitrification protocols. To date, permeation kinetics of the cryoprotectant formamide in articular cartilage have not been sufficiently explored. The objective of this study was to determine the permeation kinetics of formamide into porcine articular cartilage for application in vitrification. The permeation of dimethyl sulfoxide was first measured to validate existing methods from our previously published literature. Osteochondral dowels from dissected porcine femoral condyles were incubated in 6.5 M dimethyl sulfoxide for a designated treatment time (1 s, 1 min, 2 min, 5 min, 10 min, 15 min, 30 min, 60 min, 120 min, 180 min, 24 h) at 22 °C (N = 3). Methods were then repeated with 6.5 M formamide at one of three temperatures: 4 °C, 22 °C, 37 °C (N = 3). Following incubation, cryoprotectant efflux into a wash solution occurred, and osmolality was measured from each equilibrated wash solution. Concentrations of effluxed cryoprotectant were calculated and diffusion coefficients were determined using an analytical solution to Fick's law for axial and radial diffusion in combination with a least squares approach. The activation energy of formamide was determined from the Arrhenius equation. The diffusion coefficient (2.7-3.3 × 10-10 m2/s depending on temperature) and activation energy (0.9±0.6 kcal/mol) for formamide permeation in porcine articular cartilage were established. The determined permeation kinetics of formamide will facilitate its precise use in future articular cartilage vitrification protocols.

The effect of cryoprotectant vehicle solution on cartilage cell viability following vitrification.

Osteochondral allografts are often used to repair large articular cartilage defects to prevent or delay the onset of osteoarthritis. This approach is limited by the timely acquisition and use of allograft tissue since standard hypothermic protocols allow for a maximum storage of 4 weeks. Vitrification is a proven technique for the long-term preservation of cells and tissues, but requires careful determination of parameters to be successful, particularly for articular cartilage. One parameter that is infrequently considered is the choice of cryoprotectant vehicle solution. The aim of this study was to evaluate the impact of a subset of vehicle solutions on an established vitrification protocol for articular cartilage. These solutions were phosphate-buffered saline (PBS), Dulbecco's Modified Eagle's Medium: Nutrient Mixture F-12 (DMEM), X-VIVO, and Unisol-CV (UCV). Both the solution pH at various points throughout vitrification and the cell viability of porcine articular cartilage slices following vitrification were measured. Using randomized block ANOVA, it was found that the normalized cell viability of articular cartilage vitrified in UCV was significantly greater than that of PBS (p < 0.05) and may be greater than those of DMEM and X-VIVO (p < 0.1). There was no correlation between pH parameters and cell viability, although significant differences between calculated pH parameters were identified. These results provide information to guide the design of effective vitrification protocols for articular cartilage.

Antioxidant additives reduce reactive oxygen species production in articular cartilage during exposure to cryoprotective agents.

High concentrations of cryoprotective agents (CPA) are required during articular cartilage cryopreservation but these CPAs can be toxic to chondrocytes. Reactive oxygen species have been linked to cell death due to oxidative stress. Addition of antioxidants has shown beneficial effects on chondrocyte survival and functions after cryopreservation. The objectives of this study were to investigate (1) oxidative stress experienced by chondrocytes and (2) the effect of antioxidants on cellular reactive oxygen species production during articular cartilage exposure to high concentrations of CPAs. Porcine cartilage dowels were exposed to a multi-CPA solution supplemented with either 0.1 mg/mL chondroitin sulfate or 2000 µM ascorbic acid, at 4 °C for 180 min (N = 7). Reactive oxygen species production was measured with 5 µM dihydroethidium, a fluorescent probe that targets reactive oxygen species. The cell viability was quantified with a dual cell membrane integrity stain containing 6.25 µM Syto 13 + 9 µM propidium iodide using confocal microscopy. Supplementation of CPA solutions with chondroitin sulfate or ascorbic acid resulted in significantly lower dihydroethidium counts (p < 0.01), and a lower decrease in the percentage of viable cells (p < 0.01) compared to the CPA-treated group without additives. These results indicated that reactive oxygen species production is induced when articular cartilage is exposed to high CPA concentrations, and correlated with the amount of dead cells. Both chondroitin sulfate and ascorbic acid treatments significantly reduced reactive oxygen species production and improved chondrocyte viability when articular cartilage was exposed to high concentrations of CPAs.

Comparison of three multi-cryoprotectant loading protocols for vitrification of porcine articular cartilage.

Vitrification is a cryopreservation technique for the long-term storage of viable tissue, but the success of this technique relies on multiple factors. In 2012, our group published a working vitrification protocol for intact human articular cartilage and reported promising chondrocyte recovery after using a four-step multi-cryoprotectant (CPA) loading method that required 570 min. However, this protocol requires further optimization for clinical practice. Herein, we compared three multi-step CPA loading protocols to investigate their impact on chondrocyte recovery after vitrification of porcine articular cartilage on a bone base, including our previous four-step protocol (original: 570 min), and two shorter three-step protocols (optimized: 420 min, and minimally vitrifiable: 310 min). Four different CPAs were used including glycerol, dimethyl sulfoxide, ethylene glycol and propylene glycol. As vitrification containers, two conical tubes (50 ml and 15 ml) were evaluated for their heat transfer impact on chondrocyte recovery after vitrification. Osteochondral dowels were cored into two diameters of 10.0 mm and 6.9 mm with an approximately 10-mm thick bone base, and then allocated into the twelve experimental groups based on CPA loading protocol, osteochondral dowel size, and vitrification container size. After vitrification at -196 °C and tissue warming and CPA removal, samples in all groups were assessed for both chondrocyte viability and metabolic activity. The optimized protocol proposed based on mathematical modelling resulted in similar chondrocyte recovery to our original protocol and it was 150 min shorter. Furthermore, this study illustrated the role of CPA permeation (dowel size) and heat transfer (container size) on vitrification protocol outcome.

Using engineering models to shorten cryoprotectant loading time for the vitrification of articular cartilage.

Osteochondral allograft transplantation can treat full thickness cartilage and bone lesions in the knee and other joints, but the lack of widespread articular cartilage banking limits the quantity of cartilage available for size and contour matching. To address the limited availability of cartilage, vitrification can be used to store harvested joint tissues indefinitely. Our group's reported vitrification protocol [Biomaterials 33 (2012) 6061-6068] takes 9.5 h to load cryoprotectants into intact articular cartilage on bone and achieves high cell viability, but further optimization is needed to shorten this protocol for clinical use. Herein, we use engineering models to calculate the spatial and temporal distributions of cryoprotectant concentration, solution vitrifiability, and freezing point for each step of the 9.5-h protocol. We then incorporate the following major design choices for developing a new shorter protocol: (i) all cryoprotectant loading solution concentrations are reduced, (ii) glycerol is removed as a cryoprotectant, and (iii) an equilibration step is introduced to flatten the final cryoprotectant concentration profiles. We also use a new criterion-the spatially and temporally resolved prediction of solution vitrifiability-to assess whether a protocol will be successful instead of requiring that each cryoprotectant individually reaches a certain concentration. A total cryoprotectant loading time of 7 h is targeted, and our new 7-h protocol is predicted to achieve a level of vitrifiability comparable to the proven 9.5-h protocol throughout the cartilage thickness.

O-arm navigation versus C-arm guidance for pedicle screw placement in spine surgery: a systematic review and meta-analysis.

BACKGROUND: O-arm and C-arm are commonly used in spine surgery to guide pedicle screw placement. However, concerning the accuracy and efficiency of them, no systematical review and meta-analyses are available to help surgeons make comparisons. PURPOSES: This study aims to investigate the accuracy and efficiency of O-arm-navigated versus C-arm-guided pedicle screw placement in thoracic and lumbar spine surgery. It would help surgeons choose the optimal technique for pedicle screw placement. PATIENTS AND METHODS: A systematic review and meta-analyses were performed after searching the PubMed, Embase, and Cochrane databases to identify all studies that assessed the accuracy and efficiency of navigation coupled with O-arm and conventional C-arm fluoroscopy. RESULTS: Eight studies were finally recruited in this systematic review, all of which reported pedicle screw placement outcomes related to accuracy or efficiency in both C-arm and O-arm groups. Five studies showed higher screw insertion accuracy in the O-arm group, while one study showed no significant difference. And the pooled results also indicated that the incidence of screw misplacement in the C-arm groups is higher. Moreover, the pooled results from five studies indicated no significant difference in insertion time between C-arm and O-arm. CONCLUSIONS: Navigation coupled with O-arm imaging displayed a lower efficiency outcome in pedicle screw placement compared to conventional C-arm fluoroscopy. However, in terms of accuracy, O-arm navigation had significant advantages in accuracy over conventional C-arm fluoroscopy.

Room Temperature Versus Warm Irrigation Fluid Used for Patients Undergoing Arthroscopic Shoulder Surgery: A Systematic Review and Meta Analysis.

PURPOSE: The aim of this study was to analyze whether warm irrigation fluid could reduce postoperative adverse effects in patients undergoing arthroscopic shoulder surgery compared with room temperature irrigation fluid. DESIGN: A systematic review and meta-analysis of clinical trials was performed. METHODS: A computerized search of electronic databases was performed. The inclusion criteria were studies comparing the clinical effects of room temperature and warm irrigation fluid on patients undergoing arthroscopic shoulder surgery. FINDINGS: Warm irrigation fluid reduced the degree of core body temperature drop and the incidence of hypothermia. A statistically lower incidence of shivering also occurred in the warm irrigation fluid group. CONCLUSIONS: The use of warm irrigation fluid better maintains core body temperature and reduces incidence of shivering than room temperature irrigation fluid. Therefore, warm irrigation fluid is a better choice for arthroscopic shoulder surgery.

Evaluation of five additives to mitigate toxicity of cryoprotective agents on porcine chondrocytes.

Cryoprotective agents (CPAs) are used in cryopreservation protocols to achieve vitrification. However, the high CPA concentrations required to vitrify a tissue such as articular cartilage are a major drawback due to their cellular toxicity. Oxidation is one factor related to CPA toxicity to cells and tissues. Addition of antioxidants has proven to be beneficial to cell survival and cellular functions after cryopreservation. Investigation of additives for mitigating cellular CPA toxicity will aid in developing successful cryopreservation protocols. The current work shows that antioxidant additives can reduce the toxic effect of CPAs on porcine chondrocytes. Our findings showed that chondroitin sulphate, glucosamine, 2,3,5,6-tetramethylpyrazine and ascorbic acid improved chondrocyte cell survival after exposure to high concentrations of CPAs according to a live-dead cell viability assay. In addition, similar results were seen when additives were added during CPA removal and articular cartilage sample incubation post CPA exposure. Furthermore, we found that incubation of articular cartilage in the presence of additives for 2 days improved chondrocyte recovery compared with those incubated for 4 days. The current results indicated that the inclusion of antioxidant additives during exposure to high concentrations of CPAs is beneficial to chondrocyte survival and recovery in porcine articular cartilage and provided knowledge to improve vitrification protocols for tissue banking of articular cartilage.

The efficacy of topical tranexamic acid in total hip arthroplasty: a meta-analysis.

BACKGROUND: Topical tranexamic acid (TXA) has been shown to be effective in reducing blood loss and the need for transfusion after total knee arthroplasty. However, the effectiveness of topical TXA use in total hip arthroplasty (THA) still remains unclear. The purpose of this meta-analysis is to examine the safety and efficacy of topical use of TXA following THA. HYPOTHESIS: Topical TXA reduces blood loss and transfusion rates without increasing risk of deep vein thrombosis in patients with THA. METHODS: An electronic literature search of PubMed, Embase, the Cochrane Library, Web of Science and Chinese Biomedical Database was performed, to identify studies published before February 2015. All randomized controlled trials and cohort studies evaluating the efficacy of topical TXA during THA were included. Two independent authors identified the eligible studies, assessed their methodological quality, and extracted data. The data were using fixed-effects or random-effects models with (standard) mean differences and risk ratios for continuous and dichotomous variables, respectively. Data were analysed using RevMan 5.3 software. RESULTS: Fourteen studies encompassing 2594 patients met the inclusion criteria for our meta-analysis. Our meta-analysis indicated that when compared with the placebo group, topical use of TXA significantly reduced total blood loss (MD = -297.65 ml, 95 % CI -371.68 ml, 116.08 ml; P < 0.01), drainage loss (MD = -164.68 ml, 95 % CI -236.63 ml, -92.73 ml; P < 0.01), transfusion rate (RR = 0.26, 95 % CI 0.17, 0.40; P < 0.01) and with less of a drop in haemoglobin level (SMD = -0.66, 95 % CI -0.91, -0.41; P < 0.01) after primary THA. No significant difference in length of hospital stay (MD = -0.40, 95 % CI -0.91, 0.11; P = 0.14), deep vein thrombosis (RR = 1.19, 95 % CI 0.40, 3.57; P = 0.16) and pulmonary embolism (RR = 1.11, 95 % CI 0.11, 10.81; P = 0.21) among the study groups. CONCLUSIONS: Topical TXA could significantly reduce total blood loss, drainage loss, transfusion rates and decrease haemoglobin level following THA, without increasing risk of venous thromboembolisms.

Strong expression of paired-like homeodomain transcription factor 1 (PITX1) is associated with a favorable outcome in human osteosarcoma.

Paired-like homeodomain transcription factor 1 (PITX1) has been implicated as a tumor suppressor in various cancers. However, the biological and clinical significance of PITX1 in osteosarcoma has not been fully elucidated. Here, we studied the expression and clinical significance of PITX1 in 6 normal lower limb bone tissue specimens and 35 osteosarcoma tissue samples by immunohistochemistry. PITX1 was expressed in all normal tissues (6/6, 100 %) and in 85.7 % (30/35) of tumor tissues (P > 0.05). In addition, all normal tissue specimens showed high PITX1 expression (6/6, 100 %) while only 23.3 % (7/30) osteosarcoma tissue specimens had high PITX1 expression (P < 0.05). Patients with median overall survival (OS) >12 months had significantly higher PITX1 levels compared with those whose median OS was less than or equal to 12 months (P < 0.05 or 0.001). Furthermore, patients with lung metastasis had significantly lower PITX1 levels than patients without lung metastasis. In conclusion, PITX1 expression is downregulated in osteosarcoma and correlates with patient survival and lung metastasis.

Identification of circRNA biomarkers in osteosarcoma: An updated systematic review and meta-analysis.

Circular RNAs (circRNAs) play a crucial role in cancer development and progression. This study aimed to identify potential circRNA biomarkers for osteosarcoma. Articles published from January 2010 to September 2023 were searched across eight databases to compare circRNA expression profiles in osteosarcoma and control samples (human, animal and cell lines). Meta-analysis was conducted under a random effects model. Subgroup analysis of circRNAs in different samples and tissues was performed. Diagnostic value was evaluated using receiver operator characteristic curves. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis explored functions of circRNA host genes. A circRNA-miRNA-mRNA axis depicted the regulatory mechanism in osteosarcoma. Among 1356 circRNAs with differential expression were identified across 226 original studies, only 74 were reported in at least three published sub-studies. Meta-analysis identified 58 dysregulated circRNAs (52 upregulated and 6 downregulated). Eleven circRNAs consistently showed dysregulation in tissues and cell lines, with hsa_circ_0005721 showing potential as a circulating biomarker in osteosarcoma. Sensitivity analysis demonstrated 97 % consistency. The overall area under the curve was 0.87 (95 % CI, 0.83-0.89). GO and KEGG enrichment analyses revealed host gene involvement in cancer. The circRNA-miRNA-mRNA axis revealed the regulatory axis and interactions within osteosarcoma specifically. This study demonstrates circRNAs as potential diagnostic biomarkers for osteosarcoma. Consistently reported dysregulated circRNAs are potential biomarkers in osteosarcoma pathogenesis, with hsa_circ_0005721 as a potential circulating biomarker for diagnosis and treatment.

Mechanical Properties of Fresh, Frozen and Vitrified Articular Cartilage.

Osteochondral allograft transplantations are typically used to treat focal articular cartilage injuries where the damaged cartilage is replaced with fresh cadaveric donor grafts. Despite the notable success rate of this procedure, it is limited by fresh donor tissue availability which can only be stored for approximately 28 days after harvest. Vitrification, a form of cryopreservation, can extend the storage time of cartilage. Although it has shown to preserve chondrocyte viability, its effect on the mechanical properties of the tissue has not been thoroughly investigated. Therefore, in this study, the mechanical properties of fresh, frozen, and vitrified articular cartilage were evaluated through unconfined compression testing. Results showed that the peak modulus, equilibrium modulus, and relaxation time constants of the vitrified and control samples (tested one day after harvest) were similar and higher than the fresh (tested 21 days after harvest) and frozen samples. This demonstrated that vitrification does not adversely affect the mechanical properties of cartilage and can be used as an alternative to fresh allografts which are limited by storage time. The fresh samples also had inferior mechanical properties compared to the control samples suggesting that vitrified allografts could potentially improve clinical outcomes in addition to increasing donor tissue availability.

Chondrocyte Viability of Particulated Porcine Articular Cartilage Is Maintained in Tissue Storage After Cryoprotectant Exposure, Vitrification, and Tissue Warming.

OBJECTIVE: Vitrification of articular cartilage (AC) is a promising technique which may enable long-term tissue banking of AC allografts. We previously developed a 2-step, dual-temperature, multi-cryoprotectant agent (CPA) loading protocol to cryopreserve particulated AC (1 mm3 cubes). Furthermore, we also determined that the inclusion of ascorbic acid (AA) effectively mitigates CPA toxicity in cryopreserved AC. Prior to clinical translation, chondrocytes must remain viable after tissue re-warming and before transplantation. However, the effects of short-term hypothermic storage of particulated AC after vitrification and re-warming are not documented. This study evaluated the chondrocyte viability of post-vitrified particulated AC during a 7-day tissue storage period at 4 °C. We hypothesized that porcine particulated AC could be stored for up to 7 days after successful vitrification without significant loss of cell viability, and these results would be enhanced when cartilage is incubated in storage medium supplemented with clinical grade AA. DESIGN: Three experimental groups were examined at 5 time points: a fresh control (only incubated in medium), a vitrified - AA group, and a vitrified + AA group (N = 7). RESULTS: There was a mild decline in cell viability but both treatment groups maintained a viability of greater than 80% viable cells which is acceptable for clinical translation. CONCLUSION: We determined that particulated AC can be stored for up to 7 days after successful vitrification without a clinically significant decline in chondrocyte viability. This information can be used to guide tissue banks regarding the implementation of AC vitrification to increase cartilage allograft availability.

Effect of vitrification on mechanical properties of porcine articular cartilage.

Articular cartilage (AC) injuries do not heal primarily and large lesions progress to degenerative osteoarthritis. Osteochondral allograft transplantation is an effective surgical treatment but is limited by the lack of donor tissue availability. Fresh allografts can be stored hypothermically up to 28-45 days after which the tissue is no longer viable for transplantation. Vitrification is a method of cryopreservation with the potential to extend the storage time of AC. A specific protocol has been demonstrated to preserve high chondrocyte viability; however, its effect on various mechanical properties of the extracellular matrix (ECM) remains unknown and is the focus of this initial study. Porcine AC was subject to a defined vitrification protocol, using fresh and frozen samples as positive and negative controls, respectively; n = 20 for all three groups. Unconfined compression testing was used to assess mechanical properties of the tissue under rapid load, stress relaxation, and equilibrium conditions. The stress relaxation time constants (modeled with a 2-term Prony series) τ1 and τ2 were significantly lower for frozen (p = 0.014, p < 0.001) and vitrified (p = 0.009, p = 0.003) tissue compared to fresh, with no differences between frozen and vitrified samples (p = 0.848 and 0.105 for τ1 and τ2, respectively). These values indicate that frozen and vitrified samples relaxed more rapidly than fresh, which may suggest altered matrix composition and permeability post-treatment. These results represent the initial study in our experimental path to evaluate differences in mechanical properties of vitrified tissues.

Tensile mechanical properties of vitrified porcine menisci are superior to frozen and similar to fresh porcine menisci.

Vitrification inhibits crystallization of ice and may allow the mechanical properties of menisci to be preserved for transplantation without the damaging consequences of ice crystals formed during freezing. The primary objective of this study was to investigate the differences between fresh, frozen, and vitrified porcine lateral menisci examining tensile mechanical properties along the circumferential-peripheral, circumferential-central, longitudinal, and radial orientations. The secondary objective was to investigate the variations in the tensile mechanical properties of menisci comparing the circumferential-peripheral orientation to the three other orientations: circumferential-central, longitudinal, and radial. Quasi-static tensile testing was conducted to quantify the tensile mechanical properties of fresh, frozen and vitrified menisci. Ultimate tensile strength of frozen menisci were significantly decreased compared with fresh and vitrified menisci along three orientations: circumferential-peripheral, longitudinal, and radial. Along the circumferential-central orientation, tensile modulus of frozen menisci was significantly decreased compared with fresh menisci. The mechanical properties of vitrified menisci were comparable to fresh menisci along all four orientations. For all menisci (fresh, frozen and vitrified), ultimate tensile strength and failure strain along the circumferential-peripheral orientation were significantly increased compared with the three other orientations. Freezing was detrimental to the mechanical properties of menisci but vitrification likely avoided the negative effects of freezing thereby preserving mechanical properties that were comparable to fresh menisci. The findings of this study revealed that vitrification was superior to freezing for preserving mechanical properties of meniscal tissue; hence, vitrification is likely to be a competitive alternative to freezing for meniscal transplantation in the future.

Effectiveness of Clinical-Grade Chondroitin Sulfate and Ascorbic Acid in Mitigating Cryoprotectant Toxicity in Porcine Articular Cartilage.

High concentrations of cryoprotective agents (CPAs) are required to achieve successful vitrification of articular cartilage; however, CPA cytotoxicity causes chondrocyte death. To reduce CPA toxicity, supplementation with research-grade additives, in particular chondroitin sulfate (CS) and ascorbic acid (AA), have previously been shown to improve chondrocyte recovery and metabolic function after exposure to CPAs at hypothermic conditions. However, it is necessary to evaluate the pharmaceutical equivalent clinical grade of these additives to facilitate the supplementation of additives into future vitrification protocols, which will be designed for vitrifying human articular cartilage in tissue banks. We sought to investigate the effectiveness of clinical-grade CS, AA, and N-acetylcysteine (NAC) in mitigating toxicity to chondrocytes during CPA exposure and removal, and determine whether a combination of two additives would further improve chondrocyte viability. We hypothesized that clinical-grade additives would exert chondroprotective effects comparable to those of research-grade additives, and that this protective effect would be enhanced if two additives were combined when compared with a single additive. The results indicated that both clinical-grade and research-grade additives significantly improved cell viability (p < 0.10) compared with the negative control (CPA with no additives). CS, AA, and NAC+AA increased cell viability significantly (p < 0.10) compared with the negative control. However, NAC, NAC+CS, and CS+AA did not improve cell viability when compared with the negative control (p > 0.10). We demonstrated that supplementation with clinical-grade CS or AA significantly improved chondrocyte viability in porcine cartilage subjected to high CPA concentrations, whereas supplementation with clinical-grade NAC did not benefit chondrocyte viability. Supplementation with clinical-grade additives in CPA solutions can mitigate CPA toxicity, which will be important in translating previously developed effective protocols for the vitrification of articular cartilage to human tissue banks.