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.

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.

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.

Modeling the Simultaneous Transport of Multiple Cryoprotectants into Articular Cartilage Using a Triphasic Model.

Cryopreserving articular cartilage by vitrification can increase the availability of tissue for osteochondral allograft transplantation to treat cartilage defects. Developing well-optimized vitrification protocols can be supported by mathematical modeling to reduce the amount of trial-and-error experimentation needed. Fick's law has been used to model cryoprotectant diffusion, but it assumes ideal, dilute solution behavior, neglects water movement, and assumes diffusion of each cryoprotectant is independent of the presence of other cryoprotectants. The modified triphasic model addresses some of these shortcomings by accounting for water movement and the nonideal, nondilute nature of cryoprotectant vitrification solutions. However, it currently only exists for solutions containing a single cryoprotectant. As such, we extend the modified triphasic model to include two permeating cryoprotectants so that simultaneous diffusion occurring in vitrification protocols can be more accurately modeled. Using previously published experimental data, we determine suitable values for the fitting parameters of the new model. We then model a successful vitrification protocol for particulated cartilage cubes by calculating concentration, freezing point, vitrifiability, and strain profiles at the end of each loading step. We observe that Fick's law consistently underestimates cryoprotectant concentration throughout the cartilage compared to the modified triphasic model, leading to an underestimation of tissue vitrifiability. We additionally observe that simultaneous diffusion of cryoprotectants increases the permeation rate of each individual cryoprotectant, which Fick's law fails to consider. This suggests that using the two-cryoprotectant modified triphasic model to develop vitrification protocols could reduce excess exposure to cryoprotectants and improve preserved tissue outcomes.

Vitrified Particulated Articular Cartilage for Joint Resurfacing: A Swine Model.

BACKGROUND: The use of particulated articular cartilage for repairing cartilage defects has been well established, but its use is currently limited by the availability and short shelf life of donor cartilage. Vitrification is an ice-free cryopreservation technology at ultralow temperatures for tissue banking. An optimized vitrification protocol has been developed for particulated articular cartilage; however, the equivalency of the long-term clinical efficacy of vitrified particulated articular cartilage compared with fresh articular cartilage has not yet been determined. HYPOTHESIS: The repair effect of vitrified particulated cartilage from pigs would be equivalent to or better than that of fresh particulated cartilage stored at 4°C for 21 days. STUDY DESIGN: Controlled laboratory study. METHODS: A total of 19 pigs were randomly divided into 3 experimental groups: fresh particulated cartilage group (n = 8), vitrified particulated cartilage group (n = 8), and negative control group (no particulated cartilage in the defect; n = 3). An additional pig was used as the initial cartilage donor for the first set of surgical procedures. Pigs were euthanized after 6 months to obtain femoral condyles, and the contralateral condyle was used as the positive (no defect) control. Samples were evaluated for gross morphology using the Outerbridge and Osteoarthritis Research Society International (OARSI) scoring systems, histology (safranin O, collagen type I/II, DAPI), and chondrocyte viability using live-dead membrane integrity staining. RESULTS: There were no infections after surgery, and all 19 pigs were followed for the duration of the study. The OARSI grades for the fresh and vitrified particulated cartilage groups were 2.44 ± 1.35 and 2.00 ± 0.80, respectively, while the negative control group was graded significantly higher at 4.83 ± 0.29. Analysis of histological and fluorescent staining demonstrated that the fresh and vitrified particulated cartilage groups had equivalent regeneration within cartilage defects, with similar cell viability and densities and expression of proteoglycans and collagen type I/II. CONCLUSION: The implantation of fresh or vitrified particulated cartilage resulted in the equivalent repair of focal cartilage defects when evaluated at 6 months after surgery. CLINICAL RELEVANCE: The vitrification of particulated cartilage is a viable option for long-term storage for cartilage tissue banking and could greatly increase the availability of donor tissue for transplantation.

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.

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.

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.

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.

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.

A feature-based statistical shape model for geometric analysis of the human talus and development of universal talar prostheses.

Statistical data pertaining to anatomic variations of the human talus contain valuable information for advances in biological anthropology, diagnosis of the talar pathologies, and designing talar prostheses. A statistical shape model (SSM) can be a powerful data analysis tool for the anatomic variations of the talus. The main concern in constructing an SSM for the talus is establishing the true geometric correspondence between the talar geometries. The true correspondence complies with biological and/or mathematical homologies on the talar surfaces. In this study, we proposed a semi-automatic approach to establish a dense correspondence between talar surfaces discretized by triangular meshes. Through our approach, homologous salient surface features in the form of crest lines were detected on 49 talar surfaces. Then, the point-wise correspondence information of the crest lines was recruited to create posterior Gaussian process morphable models that non-rigidly registered the talar meshes and consequently established inter-mesh dense correspondence. The resultant correspondence perceptually represented the true correspondence as per our visual assessments. Having established the correspondence, we computed the mean shape using full generalized Procrustes analysis and constructed an SSM by means of principal component analysis. Anatomical variations and the mean shape of the talus were predicted by the SSM. As a clinically related application, we considered the mean shape and investigated the feasibility of designing universal talar prostheses. Our results suggest that the mean shape of (the shapes of) tali can be used as a scalable shape template for designing universal talar prostheses.

Time course of 3D fibrocartilage formation by expanded human meniscus fibrochondrocytes in hypoxia.

Adult human meniscus fibrocartilage is avascular and nonhealing after injury. Meniscus tissue engineering aims to replace injured meniscus with lab-grown fibrocartilage. Dynamic culture systems may be necessary to generate fibrocartilage of sufficient mechanical properties for implantation; however, the optimal static preculture conditions before initiation of dynamic culture are unknown. This study thus investigated the time course of fibrocartilage formation by human meniscus fibrochondrocytes on a three-dimensional biomaterial scaffold under various static conditions. Human meniscus fibrochondrocytes from partial meniscectomy were expanded to passage 1 (P1) or P2 (3.0 ± 0.4 and 6.5 ± 0.6 population doublings), seeded onto type I collagen scaffolds, and grown in hypoxia (HYP, 3% O2 ) or normoxia (NRX, 20% O2 ) for 3, 6, and 9 weeks. Mechanical properties were not different between P1 and P2 cell-based constructs. Mechanical properties were lower in HYP, increased continually in NRX only, and were positively correlated with glycosaminoglycan content and accumulation of hyaline cartilage-like matrix components. The most mechanically competent tissues (NRX/9 weeks) reached 1/5 of the native meniscus instantaneous compression modulus but had an increasingly hypertrophic matrix-forming phenotype. HYP consistently suppressed the hypertrophic phenotype. The results provide baselines of engineered meniscus fibrocartilage properties under static conditions, which can be used to select a preculture strategy for dynamic culture depending on the desired combination of mechanical properties, hyaline cartilage-like matrix abundance, and hypertrophic phenotype.

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.

Mechano-Hypoxia Conditioning of Engineered Human Meniscus.

Meniscus fibrochondrocytes (MFCs) experience simultaneous hypoxia and mechanical loading in the knee joint. Experimental conditions based on these aspects of the native MFC environment may have promising applications in human meniscus tissue engineering. We hypothesized that in vitro "mechano-hypoxia conditioning" with mechanical loading such as dynamic compression (DC) and cyclic hydrostatic pressure (CHP) would enhance development of human meniscus fibrocartilage extracellular matrix in vitro. MFCs from inner human meniscus surgical discards were pre-cultured on porous type I collagen scaffolds with TGF-ß3 supplementation to form baseline tissues with newly formed matrix that were used in a series of experiments. First, baseline tissues were treated with DC or CHP under hypoxia (HYP, 3% O2) for 5 days. DC was the more effective load regime in inducing gene expression changes, and combined HYP/DC enhanced gene expression of fibrocartilage precursors. The individual treatments of DC and HYP regulated thousands of genes, such as chondrogenic markers SOX5/6, in an overwhelmingly additive rather than synergistic manner. Similar baseline tissues were then treated with a short course of DC (5 vs 60 min, 10-20% vs 30-40% strain) with different pre-culture duration (3 vs 6 weeks). The longer course of loading (60 min) had diminishing returns in regulating mechano-sensitive and inflammatory genes such as c-FOS and PTGS2, suggesting that as few as 5 min of DC was adequate. There was a dose-effect in gene regulation by higher DC strains, whereas outcomes were inconsistent for different MFC donors in pre-culture durations. A final set of baseline tissues was then cultured for 3 weeks with mechano-hypoxia conditioning to assess mechanical and protein-level outcomes. There were 1.8-5.1-fold gains in the dynamic modulus relative to baseline in HYP/DC, but matrix outcomes were equal or inferior to static controls. Long-term mechano-hypoxia conditioning was effective in suppressing hypertrophic markers (e.g., COL10A1 10-fold suppression vs static/normoxia). Taken together, these results indicate that appropriately applied mechano-hypoxia conditioning can support meniscus fibrocartilage development in vitro and may be useful as a strategy for developing non-hypertrophic articular cartilage using mesenchymal stem cells.

Inability of Low Oxygen Tension to Induce Chondrogenesis in Human Infrapatellar Fat Pad Mesenchymal Stem Cells.

OBJECTIVE: Articular cartilage of the knee joint is avascular, exists under a low oxygen tension microenvironment, and does not self-heal when injured. Human infrapatellar fat pad-sourced mesenchymal stem cells (IFP-MSC) are an arthroscopically accessible source of mesenchymal stem cells (MSC) for the repair of articular cartilage defects. Human IFP-MSC exists physiologically under a low oxygen tension (i.e., 1-5%) microenvironment. Human bone marrow mesenchymal stem cells (BM-MSC) exist physiologically within a similar range of oxygen tension. A low oxygen tension of 2% spontaneously induced chondrogenesis in micromass pellets of human BM-MSC. However, this is yet to be demonstrated in human IFP-MSC or other adipose tissue-sourced MSC. In this study, we explored the potential of low oxygen tension at 2% to drive the in vitro chondrogenesis of IFP-MSC. We hypothesized that 2% O2 will induce stable chondrogenesis in human IFP-MSC without the risk of undergoing endochondral ossification at ectopic sites of implantation. METHODS: Micromass pellets of human IFP-MSC were cultured under 2% O2 or 21% O2 (normal atmosphere O2) in the presence or absence of chondrogenic medium with transforming growth factor-ß3 (TGFß3) for 3 weeks. Following in vitro chondrogenesis, the resulting pellets were implanted in immunodeficient athymic nude mice for 3 weeks. RESULTS: A low oxygen tension of 2% was unable to induce chondrogenesis in human IFP-MSC. In contrast, chondrogenic medium with TGFß3 induced in vitro chondrogenesis. All pellets were devoid of any evidence of undergoing endochondral ossification after subcutaneous implantation in athymic mice.

Clinical Use of Talar Prostheses.

¼: The blood supply to the talus is vulnerable to damage, making the talus susceptible to osteonecrosis, with limited treatment options. ¼: Talar bone replacement has been investigated as a treatment option to preserve ankle function and maintain limb length. ¼: Successful talar bone replacements have been performed for the past >35 years, with variations in design, methods of fixation, materials, and manufacturing techniques. ¼: The designs of talar prostheses range from custom-made partial (talar body) or total prostheses to prefabricated universal (non-custom-made) prostheses. ¼: Total talar prostheses have been demonstrated to function better than partial talar prostheses; however, there is a need for long-term studies regarding custom-made total talar prostheses and prefabricated universal talar prostheses in order to determine their long-term effectiveness.

Vitrification of particulated articular cartilage via calculated protocols.

Preserving viable articular cartilage is a promising approach to address the shortage of graft tissue and enable the clinical repair of articular cartilage defects in articulating joints, such as the knee, ankle, and hip. In this study, we developed two 2-step, dual-temperature, multicryoprotectant loading protocols to cryopreserve particulated articular cartilage (cubes ~1 mm3 in size) using a mathematical approach, and we experimentally measured chondrocyte viability, metabolic activity, cell migration, and matrix productivity after implementing the designed loading protocols, vitrification, and warming. We demonstrated that porcine and human articular cartilage cubes can be successfully vitrified and rewarmed, maintaining high cell viability and excellent cellular function. The vitrified particulated articular cartilage was stored for a period of 6 months with no significant deterioration in chondrocyte viability and functionality. Our approach enables high-quality long-term storage of viable articular cartilage that can alleviate the shortage of grafts for use in clinically repairing articular cartilage defects.

Engineered human meniscus' matrix-forming phenotype is unaffected by low strain dynamic compression under hypoxic conditions.

Low oxygen and mechanical loading may play roles in regulating the fibrocartilaginous phenotype of the human inner meniscus, but their combination in engineered tissues remains unstudied. Here, we investigated how continuous low oxygen ("hypoxia") combined with dynamic compression would affect the fibrocartilaginous "inner meniscus-like" matrix-forming phenotype of human meniscus fibrochondrocytes (MFCs) in a porous type I collagen scaffold. Freshly-seeded MFC scaffolds were cultured for 4 weeks in either 3 or 20% O2 or pre-cultured for 2 weeks in 3% O2 and then dynamically compressed for 2 weeks (10% strain, 1 Hz, 1 h/day, 5 days/week), all with or without TGF-ß3 supplementation. TGF-ß3 supplementation was found necessary to induce matrix formation by MFCs in the collagen scaffold regardless of oxygen tension and application of the dynamic compression loading regime. Neither hypoxia under static culture nor hypoxia combined with dynamic compression had significant effects on expression of specific protein and mRNA markers for the fibrocartilaginous matrix-forming phenotype. Mechanical properties significantly increased over the two-week loading period but were not different between static and dynamic-loaded tissues after the loading period. These findings indicate that 3% O2 applied immediately after scaffold seeding and dynamic compression to 10% strain do not affect the fibrocartilaginous matrix-forming phenotype of human MFCs in this type I collagen scaffold. It is possible that a delayed hypoxia treatment and an optimized pre-culture period and loading regime combination would have led to different outcomes.

Correction: Engineered human meniscus' matrix-forming phenotype is unaffected by low strain dynamic compression under hypoxic conditions.

[This corrects the article DOI: 10.1371/journal.pone.0248292.].

Human engineered meniscus transcriptome after short-term combined hypoxia and dynamic compression.

This study investigates the transcriptome response of meniscus fibrochondrocytes (MFCs) to the low oxygen and mechanical loading signals experienced in the knee joint using a model system. We hypothesized that short term exposure to the combined treatment would promote a matrix-forming phenotype supportive of inner meniscus tissue formation. Human MFCs on a collagen scaffold were stimulated to form fibrocartilage over 6 weeks under normoxic (NRX, 20% O2) conditions with supplemented TGF-ß3. Tissues experienced a delayed 24h hypoxia treatment (HYP, 3% O2) and then 5 min of dynamic compression (DC) between 30 and 40% strain. Delayed HYP induced an anabolic and anti-catabolic expression profile for hyaline cartilage matrix markers, while DC induced an inflammatory matrix remodeling response along with upregulation of both SOX9 and COL1A1. There were 41 genes regulated by both HYP and DC. Overall, the combined treatment supported a unique gene expression profile favouring the hyaline cartilage aspect of inner meniscus matrix and matrix remodeling.