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. 2015 Feb;21(3-4):829-39.
doi: 10.1089/ten.TEA.2014.0250. Epub 2015 Jan 9.

Engineered Microporosity: Enhancing the Early Regenerative Potential of Decellularized Temporomandibular Joint Discs

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Free PMC article

Engineered Microporosity: Enhancing the Early Regenerative Potential of Decellularized Temporomandibular Joint Discs

Cassandra M Juran et al. Tissue Eng Part A. .
Free PMC article

Abstract

The temporomandibular joint (TMJ) disc is susceptible to numerous pathologies that may lead to structural degradation and jaw dysfunction. The limited treatment options and debilitating nature of severe temporomandibular disorders has been the primary driving force for the introduction and development of TMJ disc tissue engineering as an approach to alleviate this important clinical issue. This study aimed to evaluate the efficacy of laser micropatterning (LMP) ex vivo-derived TMJ disc scaffolds to enhance cellular integration, a major limitation to the development of whole tissue implant technology. LMP was incorporated into the decellularized extracellular matrix scaffold structure using a 40 W CO2 laser ablation system to drill an 8×16 pattern with a bore diameter of 120 μm through the scaffold thickness. Disc scaffolds were seeded with human neonatal-derived umbilical cord mesenchymal stem cells differentiated into chondrocytes at a density of 900 cells per mm(2) and then assessed on days 1, 7, 14, and 21 of culture. Results derived from histology, PicoGreen DNA quantification, and cellular metabolism assays indicate that the LMP scaffolds improve cellular remodeling compared to the unworked scaffold over the 21-day culture period. Mechanical analysis further supports the use of the LMP showing the compressive properties of the LMP constructs closely represent native disc mechanics. The addition of an artificial path of infiltration by LMP culminated in improved chondrocyte adhesion, dispersion, and migration after extended culture aiding in recapitulating the native TMJ disc characteristics.

Figures

<b>FIG. 1.</b>
FIG. 1.
Temporomandibular joint (TMJ) anatomy and joint articulation. (A, B) The articulation of the mandibular condyle within the infratemporal fossa while (C) is an expanded illustration of the major skeletal connections of the TMJ and the articulating disc. (1) Articular fossa, (2) mandibular condyle, (3) articular eminence, and (4) TMJ fibrocartilage disc.
<b>FIG. 2.</b>
FIG. 2.
Macrostructure and histological evaluation of detergent decellularization and lyophilization of TMJ disc. The native tissue has well-defined regional segmentation and very ordered extracellular matrix (ECM) fibril alignment, including well-defined collagen bundles (A, B). The sodium dodecyl sulfate (SDS) decellularized disc macroscopically shows no pigmentation, and the microstructure has no evident cellular remnants; however, the ECM fibrils are compacted and irregularly aligned (C, D). After the acellular scaffold is freeze-dried and rehydrated, the scaffold regains some of the native tissues fibril alignment's isometric quality and the individual ECM fibers have regained their collagen bundle conformation (E, F).
<b>FIG. 3.</b>
FIG. 3.
Controllable uniformity of laser micropatterning (LMP) the TMJ disc scaffold. Scanning electron micrographs (SEMs) of the LMP TMJ disc illustrate that while there are minor (within experimental tolerances±50 μm) differences between hole diameters due to surface irregularities of the disc, the holes are uniformly spaced and traverse the thickness of the scaffold. (A) The pattern ablation into the whole TMJ disc tissue including an inset illustrating the LMP punch removed from the central region of the disc used for further evaluation. (B, C) Examination of the LMP holes via SEM. The SEMs illustrate that the surface of the TMJ disc is irregular, which causes cusping and burn rippling at the initial laser–tissue interaction. SEM of a single LMP hole ablated through the scaffold is shown in (D).
<b>FIG. 4.</b>
FIG. 4.
Mechanical energy dissipation consequence of the scaffold processing technique. The mechanical ability of the TMJ disc at each stage of the scaffold processing technique was evaluated by cyclic compressive testing. Ten percent compressive strain was applied by a flat indenter and the resulting force was measured. From the force and surface area measured, the stress was calculated and is presented in this hysteresis. Higher stress and hysteresis are seen in the SDS decellularized tissue due to ECM protein conformational disruption by the highly ionic SDS molecule. Both stress and hysteresis recoup toward native properties after freeze-drying and rehydration. LMP of 120 μm pores increases stress marginally but does no increase hysteresis.
<b>FIG. 5.</b>
FIG. 5.
Cell attachment over initial 24 h. Scaffolds were seeded with a density of 900 cells per mm2 (∼25,500 cells total/scaffold) and incubated at 5% CO2 and 37°C for 10 min to 24 h and then evaluated for cellular concentration using the Quanti-iT PicoGreen assay. After 10 min, the LMP construct retained greater than 60% of cell seeding concentration and more than 80% of those cells remained adhered after the 24 h of incubation. The non-LMP scaffold, however, only retains about 45% of the 10 min cell density (n=9, *p<0.05, **p<0.01). Data for the SDS treated (null DNA criteria) are included and demonstrated statistical difference from all seeded samples.
<b>FIG. 6.</b>
FIG. 6.
Regional DNA quantification and cell seeding density as a function of thickness. (A) Cell adhesion was further evaluated by conducting DNA quantification with the Quanti-iT PicoGreen assay regionally from the superior surface through the scaffolds thickness at 2 h after seeding (B) and 24 h after seeding (C). The analysis reveals that both the LMP and the non-LMP scaffolds retain a large cell population from seeding; however, the LMP scaffold has cell infiltration into the mid region of the tissue, identified in illustration (A), while the non-LMP scaffold has no cell infiltration past the surface regions (n=9, *p<0.05, **p<0.01).
<b>FIG. 7.</b>
FIG. 7.
Cellular attachment to the surface layer and through the cross section of the TMJ disc construct. Calcein-AM florescence staining of the top surface of the LMP (A) and non-LMP (B) scaffolds imaged 24 h after seeding shows that living cells have adhered and organized into a peripheral layer. DAPI staining identifies the nuclei of cells in cross-sectional histological slides taken at the centerline (3 mm from the edge) of both scaffolds (C, D). The cross-sectional images clearly illustrate that the LMP scaffold have greater cellular adhesion because of the additional surface area the cells are exposed to, while the non-LMP scaffold has cell adhesion only at the periphery of the cross-sectioned sample. Color images available online at www.liebertpub.com/tea
<b>FIG. 8.</b>
FIG. 8.
Cellular proliferation and metabolism and mechanical characterization over the culture period. (A) Both the LMP and the non-LMP scaffolds show increase in cell number (bar graph) and cellular metabolism (dashed lines) during the culture period with the LMP scaffold exhibiting greater increases in both, likely due to higher initial cell adhesion because of the laser holes. (B) Compressive mechanical testing reveals that the compressive modulus of both the LMP and the non-LMP scaffolds at day 1 are comparable to the native disc properties. By day 7 of culture, the compressive properties of the LMP scaffold have significantly increased indicating a stiffening of the matrix; however, at day 14 and 21, the mechanical properties begin to decrease linearly similar to the non-LMP scaffold (n=9, *p<0.05).
<b>FIG. 9.</b>
FIG. 9.
Histological evaluation of culture at day 21. (A, D) DAPI/rhodamine phaloidin staining, illustrating that cellular presence within the constructs. (B, E) Hematoxylin and eosin (H&E) imaging at the same magnification as the florescence images and (C, F) are higher magnification sections of (B, E), respectively. (A–C) Cellular infiltration has spread between the LMP holes and has created a relatively uniform cell population, while (D, E) show that the non-LMP construct has only a dense layer of cells at the construct periphery and (F) shows no recognizable cells in the interior of the non-LMP construct. Sampling was taken from 500 μm from the superior surface of the samples for comparative consistency. Color images available online at www.liebertpub.com/tea

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