Viable Vitreous Grafts of Whole Porcine Menisci for Transplant in the Knee and Temporomandibular Joints.

The shortage of suitable donor meniscus grafts from the knee and temporomandibular joint (TMJ) impedes treatments for millions of patients. Vitrification offers a promising solution by transitioning these tissues into a vitreous state at cryogenic temperatures, protecting them from ice crystal damage using high concentrations of cryoprotectant agents (CPAs). However, vitrification's success is hindered for larger tissues (>3 mL) due to challenges in CPA penetration. Dense avascular meniscus tissues require extended CPA exposure for adequate penetration; however, prolonged exposure becomes cytotoxic. Balancing penetration and reducing cell toxicity is required. To overcome this hurdle, a simulation-based optimization approach is developed by combining computational modeling with microcomputed tomography (µCT) imaging to predict 3D CPA distributions within tissues over time accurately. This approach minimizes CPA exposure time, resulting in 85% viability in 4-mL meniscal specimens, 70% in 10-mL whole knee menisci, and 85% in 15-mL whole TMJ menisci (i.e., TMJ disc) post-vitrification, outperforming slow-freezing methods (20%-40%), in a pig model. The extracellular matrix (ECM) structure and biomechanical strength of vitreous tissues remain largely intact. Vitreous meniscus grafts demonstrate clinical-level viability (≥70%), closely resembling the material properties of native tissues, with long-term availability for transplantation. The enhanced vitrification technology opens new possibilities for other avascular grafts.

Nanowarming and ice-free cryopreservation of large sized, intact porcine articular cartilage.

Successful organ or tissue long-term preservation would revolutionize biomedicine. Cartilage cryopreservation enables prolonged shelf life of articular cartilage, posing the prospect to broaden the implementation of promising osteochondral allograft (OCA) transplantation for cartilage repair. However, cryopreserved large sized cartilage cannot be successfully warmed with the conventional convection warming approach due to its limited warming rate, blocking its clinical potential. Here, we develope a nanowarming and ice-free cryopreservation method for large sized, intact articular cartilage preservation. Our method achieves a heating rate of 76.8 °C min-1, over one order of magnitude higher than convection warming (4.8 °C min-1). Using systematic cell and tissue level tests, we demonstrate the superior performance of our method in preserving large cartilage. A depth-dependent preservation manner is also observed and recapitulated through magnetic resonance imaging and computational modeling. Finally, we show that the delivery of nanoparticles to the OCA bone side could be a feasible direction for further optimization of our method. This study pioneers the application of nanowarming and ice-free cryopreservation for large articular cartilage and provides valuable insights for future technique development, paving the way for clinical applications of cryopreserved cartilage.

A noninvasive fluorescence imaging-based platform measures 3D anisotropic extracellular diffusion.

Diffusion is a major molecular transport mechanism in biological systems. Quantifying direction-dependent (i.e., anisotropic) diffusion is vitally important to depicting how the three-dimensional (3D) tissue structure and composition affect the biochemical environment, and thus define tissue functions. However, a tool for noninvasively measuring the 3D anisotropic extracellular diffusion of biorelevant molecules is not yet available. Here, we present light-sheet imaging-based Fourier transform fluorescence recovery after photobleaching (LiFT-FRAP), which noninvasively determines 3D diffusion tensors of various biomolecules with diffusivities up to 51 µm2 s-1, reaching the physiological diffusivity range in most biological systems. Using cornea as an example, LiFT-FRAP reveals fundamental limitations of current invasive two-dimensional diffusion measurements, which have drawn controversial conclusions on extracellular diffusion in healthy and clinically treated tissues. Moreover, LiFT-FRAP demonstrates that tissue structural or compositional changes caused by diseases or scaffold fabrication yield direction-dependent diffusion changes. These results demonstrate LiFT-FRAP as a powerful platform technology for studying disease mechanisms, advancing clinical outcomes, and improving tissue engineering.

Depth- and direction-dependent changes in solute transport following cross-linking with riboflavin and UVA light in ex vivo porcine cornea.

Diffusion is an important mechanism of transport for nutrients and drugs throughout the avascular corneal stroma. The purpose of this study was to investigate the depth- and direction-dependent changes in stromal transport properties and their relationship to changes in collagen structure following ultraviolet A (UVA)-riboflavin induced corneal collagen cross-linking (CXL). After cross-linking in ex vivo porcine eyes, fluorescence recovery after photobleaching (FRAP) was performed to measure fluorescein diffusion in the nasal-temporal (NT) and anterior-posterior (AP) directions at corneal depths of 100, 200, and 300 µm. Second harmonic generation (SHG) imaging was also performed at these three corneal depths to quantify fiber alignment. For additional confirmation, an electrical conductivity method was employed to quantify ion permeability in the AP direction in corneal buttons and immunohistochemistry (IHC) was used to image collagen structure. Cross-linked corneas were compared to a control treatment that received the riboflavin solution without UVA light (SHAM). The results of FRAP revealed that fluorescein diffusivity decreased from 23.39 ± 11.60 µm2/s in the SHAM group to 19.87 ± 10.10 µm2/s in the CXL group. This change was dependent on depth and direction: the decrease was more pronounced in the 100 µm depth (P = 0.0005) and AP direction (P = 0.001) when compared to the effect in deeper locations and in the NT direction, respectively. Conductivity experiments confirmed a decrease in solute transport in the AP direction (P < 0.0001). FRAP also detected diffusional anisotropy in the porcine cornea: the fluorescein diffusivity in the NT direction was higher than the diffusivity in the AP direction. This anisotropy was increased following CXL treatment. Both SHG and IHC revealed a qualitative decrease in collagen crimping following CXL. Analysis of SHG images revealed an increase in coherency in the anterior 200 µm of CXL treated corneas when compared to SHAM treated corneas (P < 0.01). In conclusion, CXL results in a decrease in stromal solute transport, and this decrease is concentrated in the most anterior region and AP direction. Solute transport in the porcine cornea is anisotropic, and an increase in anisotropy with CXL may be explained by a decrease in collagen crimping.

The Impact of Heat Treatment on Porcine Heart Valve Leaflets.

The purpose of this study was to determine the impact of elevated temperature exposure in tissue banking on soft tissues. A secondary objective was to determine the relative ability of various assays to detect changes in soft tissues due to temperature deviations. Porcine pulmonary heart valve leaflets exposed to 37 °C were compared with those incubated at 52 and 67 °C for 10, 30 and 100 min. The analytical methods consisted of (1) viability assessment using the resazurin assay, (2) collagen content using the Sircol assay, and (3) permeability assessment using an electrical conductivity assay. Additionally, histology and two photon microscopy were used to reveal mechanisms of cell and tissue damage. Viability, collagen content, and permeability all decreased following heat treatment. In terms of statistical significance with respect to treatment temperature, cell viability was most affected (p < 0.0001), followed by permeability (p < 0.0001), and then collagen content (p = 0.13). After heat treatment, histology indicated increased apoptosis and two photon microscopy revealed a decrease in collagen fiber organization and an increase in elastin density. These results suggest that measures of cell viability would be best for assessing tissues where the cells are alive and that permeability may be best where cell viability is not intentionally maintained.

Tensile biomechanical properties of human temporomandibular joint disc: Effects of direction, region and sex.

Approximately 30% of temporomandibular joint (TMJ) disorders include degenerative changes to the articular disc, with sex-specific differences in prevalence and severity. Limited tensile biomechanical properties of human TMJ discs have been reported. Stress relaxation tests were conducted on TMJ disc specimens harvested bilaterally from six males and six females (68.9±7.9 years), with step-strain increments of 5%, 10%, 15%, 20% and 30%, at 1% strain-per-second. Stress versus strain plots were constructed, and Young׳s Modulus, Instantaneous Modulus and Relaxed Modulus were determined. The effects of direction, region, and sex were examined. Regional effects were significant (p<0.01) for Young׳s Modulus and Instantaneous Modulus. Anteroposteriorly, the central region was significantly stiffer than medial and lateral regions. Mediolaterally, the posterior region was significantly stiffer than central and anterior regions. In the central region, anteroposteriorly directed specimens were significantly stiffer compared to mediolateral specimens (p<0.04). TMJ disc stiffness, indicated by Young׳s Modulus and Instantaneous Modulus, was higher in directions corresponding to high fiber alignment. Additionally, human TMJ discs were stiffer for females compared to males, with higher Young׳s Modulus and Instantaneous Modulus, and female TMJ discs relaxed less. However, sex effects were not statistically significant. Using second-harmonic generation microscopy, regional collagen fiber organization was identified as a potentially significant factor in determining the biomechanical properties for any combination of direction and region. These findings establish structure-function relationships between collagen fiber direction and organization with biomechanical response to tensile loading, and may provide insights into the prevalence of TMJ disorders among women.

Comparison and evaluation of biomechanical, electrical, and biological methods for assessment of damage to tissue collagen.

In regard to evaluating tissue banking methods used to preserve or otherwise treat (process) soft allograft tissue, current tests may not be sufficiently sensitive to detect potential damage inflicted before, during, and after processing. Using controlled parameters, we aim to examine the sensitivity of specific biomechanical, electrical, and biological tests in detecting mild damage to collagen. Fresh porcine pulmonary heart valves were treated with an enzyme, collagenase, and incubated using various times. Controls received no incubation. All valves were cryopreserved and stored at -135 °C until being rewarmed for evaluation using biomechanical, permeability, and cell viability tests. Statistically significant time dependent changes in leaflet ultimate stress, (p = 0.006), permeability (p = 0.01), and viability (p ≤ 0.02, four different days of culture) were found between heart valves subjected to 0-15 min of collagenase treatment (ANOVA). However, no statistical significance was found between the tensile modulus of treated and untreated valves (p = 0.07). Furthermore, the trends of decreasing and increasing ultimate stress and viability, respectively, were somewhat inconsistent across treatment times. These results suggest that permeability tests may offer a sensitive, quantitative assay to complement traditional biomechanical and viability tests in evaluating processing methods used for soft tissue allografts, or when making changes to current validated methods. Multiple test evaluation may also offer insight into the mechanism of potential tissue damage such as, as is the case here, reduced collagen content and increased tissue porosity.

Decreased Mechanical Strength and Collagen Content in SPARC-Null Periodontal Ligament Is Reversed by Inhibition of Transglutaminase Activity.

The periodontal ligament (PDL) is a critical tissue that provides a physical link between the mineralized outer layer of the tooth and the alveolar bone. The PDL is composed primarily of nonmineralized fibrillar collagens. Expression of secreted protein acidic and rich in cysteine (SPARC/osteonectin), a collagen-binding matricellular protein, has been shown to be essential for collagen homeostasis in PDL. In the absence of SPARC, PDL collagen fibers are smaller and less dense than fibers that constitute WT PDL. The aim of this study was to identify cellular mechanisms by which SPARC affected collagen fiber assembly and morphology in PDL. Cross-linking of fibrillar collagens is one parameter that is known to affect insoluble collagen incorporation and fiber morphology. Herein, the reduction in collagen fiber size and quantity in the absence of SPARC expression was shown to result in a PDL with reduced molar extraction force in comparison to that of WT mice (C57Bl/6J). Furthermore, an increase in transglutaminase activity was found in SPARC-null PDL by biochemical analyses that was supported by immunohistochemical results. Specifically, collagen I was identified as a substrate for transglutaminase in PDL and transglutaminase activity on collagen I was found to be greater in SPARC-null tissues in comparison to WT. Strikingly, inhibition of transglutaminase activity in SPARC-null PDL resulted in increases in both collagen fiber thickness and in collagen content, whereas transglutaminase inhibitors injected into WT mice resulted in increases in collagen fiber thickness only. Furthermore, PDL treated with transglutaminase inhibitors exhibited increases in molar extraction force in WT and in SPARC-null mice. Thus, SPARC is proposed to act as a critical regulator of transglutaminase activity on collagen I with implications for mechanical strength of tissues.

Corneal cross-linking: engineering a predictable model.

Corneal collagen cross-linking (CXL) with riboflavin and ultraviolet-A (UVA) light has become a viable treatment for keratoconus. In cases in which corneal transplant may have previously been a patient's primary treatment option, the results of CXL have varied from decreased progression of the disease to marked regression characterized by improvement in visual acuity. In addition, changes to the original protocol have been tested that include leaving the epithelium intact and increasing the UVA intensity while decreasing the exposure time. The variation in results and protocols underscores the need for a greater understanding of the procedure and its effects. Ideally, a complete definition of the effects of CXL will lead to patient-specific treatment through highly controlled delivery methods of riboflavin and UVA light and complete mathematical models for predicting the final shape and refractive effect of the cornea. Thus, in this review, we aimed to describe the current techniques for measuring the effect of CXL, with a focus on material property changes, while highlighting the challenge of engineering a predictable mathematical model of the procedure and the resulting clinical outcome.

Precisely Assembled Nanofiber Arrays as a Platform to Engineer Aligned Cell Sheets for Biofabrication.

A hybrid cell sheet engineering approach was developed using ultra-thin nanofiber arrays to host the formation of composite nanofiber/cell sheets. It was found that confluent aligned cell sheets could grow on uniaxially-aligned and crisscrossed nanofiber arrays with extremely low fiber densities. The porosity of the nanofiber sheets was sufficient to allow aligned linear myotube formation from differentiated myoblasts on both sides of the nanofiber sheets, in spite of single-side cell seeding. The nanofiber content of the composite cell sheets is minimized to reduce the hindrance to cell migration, cell-cell contacts, mass transport, as well as the foreign body response or inflammatory response associated with the biomaterial. Even at extremely low densities, the nanofiber component significantly enhanced the stability and mechanical properties of the composite cell sheets. In addition, the aligned nanofiber arrays imparted excellent handling properties to the composite cell sheets, which allowed easy processing into more complex, thick 3D structures of higher hierarchy. Aligned nanofiber array-based composite cell sheet engineering combines several advantages of material-free cell sheet engineering and polymer scaffold-based cell sheet engineering; and it represents a new direction in aligned cell sheet engineering for a multitude of tissue engineering applications.