Gregory J. Wright

Allogeneic heart valve storage above the glass transition at -80°C.

BACKGROUND Cryopreserved allogeneic heart valves are usually stored and transported below -135°C; however, such methods require expensive equipment for both storage and transportation. METHODS In this study, vitrified porcine aortic valves were stored on either side of the cryoprotectant formulations glass transition temperature (-119°C) at -80°C and -135°C, using a newly formulated vitrification solution (VS83) consisting of a combination of 4.65M dimethyl sulfoxide, 4.65M formamide, and 3.30M 1,2-propanediol. Three groups of valves were studied: (1) fresh; (2) VS83-preserved, stored at -80°C; and (3) VS83-preserved, stored at -135°C. RESULTS Using the VS83 cryoprotectant concentration formulation, cracking was not observed during valve storage. No ice-related events were detectable during 5°C rewarming by differential scanning calorimetry. All cryopreserved tissue samples demonstrated significantly less viability than fresh samples (p<0.01). No significant viability differences were observed between the VS83-preserved groups stored at -80°C and -135°C. Material testing did not reveal any significant differences among the three test groups. Multiphoton imaging of VS83-preserved heart valves stored at -80°C and -135°C demonstrated similar collagen and elastin structures. CONCLUSIONS These results indicate that VS83-preserved heart valves can be stored and transported at temperatures in the vicinity of -80°C with retention of extracellular matrix integrity and material properties. The VS83 preservation of heart valves at -80°C without the need for liquid nitrogen should result in both decreased manufacturing costs and reduced employee safety hazards. Moreover, it is anticipated that low cell viability may result in less immunogenicity in vivo.

Effect of Mechanical Loading on Electrical Conductivity in Porcine TMJ Discs

The objective of this study was to examine the impact of mechanical loading on solute transport in porcine temporomandibular joint (TMJ) discs using the electrical conductivity method. The electrical conductivity, as well as ion diffusivity, of TMJ discs was determined under confined compression with 3 strains in 5 disc regions. The average electrical conductivity over the 5 regions (mean ± SD) at 0% strain was 3.10 ± 0.68 mS/cm, decreased to 2.76 ± 0.58 mS/cm (-11.0%) at 10% strain, and 2.38 ± 0.55 mS/cm (-22.2%) at 20% compressive strain. Correspondingly, the average relative ion diffusivity (mean ± SD) at 0% strain was 0.273 ± 0.055, decreased to 0.253 ± 0.048 (-7.3%) at 10% strain, and 0.231 ± 0.048 (-15.4%) at 20% compressive strain. These results indicated that compressive strain impeded solute transport in the TMJ disc. Furthermore, our results showed that the transport properties of TMJ discs were region-dependent. The electrical conductivity and ion diffusivity in the anterior region were significantly higher than in the posterior region. This regional difference is likely due to the significant differences of tissue hydration between these 2 regions. This study provides important insight into the electrical and solute transport behaviors in TMJ discs under mechanical loading and aids in the understanding of TMJ pathophysiology related to tissue nutrition.

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.

Impact of Hypothermia upon Chondrocyte Viability and Cartilage Matrix Permeability after 1 Month of Refrigerated Storage

Background: The purpose of this research was to assess the extracellular matrix and chondrocytes of articular cartilage during refrigerated storage and to determine whether changes could be detected in the time frame that cartilage is stored for clinical use. Methods: Porcine cartilage was stored as either bisected femoral heads with bone attached or plugs without the underlying bone in culture medium with fetal bovine serum for 1 month at 4 °C. Metabolic activity was tested using a resazurin reduction method on intact tissue and viable cell recovery after enzymatic tissue digestion at each time point. Cartilage plug permeability was evaluated by measuring electrical conductivity. Results: Storage in culture medium provided good cartilage viability and metabolic function for 7 days; however, significant changes were observed in femoral heads (p < 0.05). All mean chondrocyte assessment values were <30% of fresh controls at 28 days. Cartilage plugs tended to perform better after 7 days of storage than the femoral heads and retained significantly higher metabolic activity (mean = 94.5% vs. 70.5%; p < 0.05). Cartilage plugs demonstrated consistent changes in electrical conductivity after 28 days of storage (p < 0.05). Conclusion: Refrigerated storage of cartilage results in both loss of chondrocyte viability and matrix permeability.

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.

The effects of oxygen level and glucose concentration on the metabolism of porcine TMJ disc cells

OBJECTIVE To determine the combined effect of oxygen level and glucose concentration on cell viability, ATP production, and matrix synthesis of temporomandibular joint (TMJ) disc cells. DESIGN TMJ disc cells were isolated from pigs aged 6-8 months and cultured in a monolayer. Cell cultures were preconditioned for 48 h with 0, 1.5, 5, or 25 mM glucose DMEM under 1%, 5%, 10%, or 21% O2 level, respectively. The cell viability was measured using the WST-1 assay. ATP production was determined using the Luciferin-Luciferase assay. Collagen and proteoglycan synthesis were determined by measuring the incorporation of [2, 3-(3)H] proline and [(35)S] sulfate into the cells, respectively. RESULTS TMJ disc cell viability significantly decreased (P < 0.0001) without glucose. With glucose present, decreased oxygen levels significantly increased viability (P < 0.0001), while a decrease in glucose concentration significantly decreased viability (P < 0.0001). With glucose present, decreasing oxygen levels significantly reduced ATP production (P < 0.0001) and matrix synthesis (P < 0.0001). A decreased glucose concentration significantly decreased collagen synthesis (P < 0.0001). The interaction between glucose and oxygen was significant in regards to cell viability (P < 0.0001), ATP production (P = 0.00015), and collagen (P = 0.0002) and proteoglycan synthesis (P < 0.0001). CONCLUSIONS Although both glucose and oxygen are important, glucose is the limiting nutrient for TMJ disc cell survival. At low oxygen levels, the production of ATP, collagen, and proteoglycan are severely inhibited. These results suggest that steeper nutrient gradients may exist in the TMJ disc and it may be vulnerable to pathological events that impede nutrient supply.

Impact of Storage Solution Formulation during Refrigerated Storage upon Chondrocyte Viability and Cartilage Matrix

Various preservation solutions have been evaluated for longer hypothermic cartilage storage for tissue transplantation; however, the results are mixed. This research was carried out to determine whether phosphate-buffered saline (PBS) or organ preservation solutions would preserve both the extracellular matrix and chondrocytes of articular cartilage better than culture medium during refrigerated storage in the time frame that cartilage is stored for clinical use. Porcine cartilage plugs were stored, without the underlying bone, in culture medium with and without fetal bovine serum (FBS), PBS, Belzers and Unisol solutions for 1 month at 4°C. Metabolic activity was tested using a resazurin reduction method, and matrix permeability was evaluated by measuring electrical conductivity. Storage in culture medium with 10% FBS was shown to provide good cartilage metabolic function for 7 days, decreasing to about 36% after 1 month of storage. There was no significant difference between samples stored in culture medium with and without FBS after 1 month of storage (p = 0.5005). Refrigerated storage of cartilage in PBS and two different solutions (Belzers and Unisol) designed for optimal refrigerated tissue and organ storage results in loss of chondrocyte function and retention of matrix permeability. In contrast, the opposite, namely significantly better retention of chondrocyte function and loss of matrix permeability, was observed with culture medium. Future research should be focused on combining retention of chondrocyte function and matrix permeability by storage solution formulation.

Structure-Function Relationships of Temporomandibular Retrodiscal Tissue:

It is estimated that 2% to 4% of the US population will seek treatment for temporomandibular joint (TMJ) symptoms, typically occurring with anterior disc displacement. The temporomandibular retrodiscal tissue (RDT) has been postulated to restrict pathologic disc displacement. To elucidate RDT function, understanding regional RDT biomechanics and ultrastructure is required. No prior biomechanical analysis has determined regional variations in RDT properties or associated biomechanical outcomes with regional variations in collagen and elastin organization. The purpose of this study was to determine direction- and region-dependent tensile biomechanical characteristics and regional fibrillar arrangement of porcine RDT. Incremental stress relaxation experiments were performed on 20 porcine RDT specimens, with strain increments from 5% to 50%, a ramp-strain rate of 2% per second, and relaxation periods of 2.5 min. Tensile characteristics were determined between temporal and condylar regions and anteroposterior and mediolateral directions. RDT preparations were imaged using second-harmonic generation (SHG) microscopy for both collagen and elastin. Young’s modulus showed significant differences by region (P < 0.001) and strain (P < 0.001). Young’s modulus was <1 MPa from 5% to 20% strain, before increasing from 20% to 50% strain to a maximum of 2.9 MPa. Young’s modulus trended higher in the temporal region and mediolateral direction. Instantaneous and relaxed moduli showed no significant difference by region or direction. Collagen arrangement was most organized near the disc boundary, with disorganization increasing posteriorly. Elastin was present at the disc boundary and RDT mid-body. Porcine RDT demonstrated region- and strain-dependent variations in tensile moduli, associated with regional differences in collagen and elastin. The small tensile moduli suggest that the RDT is not resistive to pathologic disc displacement. Further biomechanical analysis of the RDT is required to fully define RDT functional roles. Understanding regional variations in tissue stiffness and ultrastructure for TMJ components is critical to understanding joint function and for the long-term goal of improving TMJ disorder treatment strategies.