Characterization of natural, decellularized and reseeded porcine tooth bud matrices

Abstract

Dental tissue engineering efforts have yet to identify scaffolds that instruct the formation of bioengineered teeth of predetermined size and shape. Here we investigated whether extracellular matrix (ECM) molecules present in natural tooth scaffolds can provide insight on how to achieve this goal. We describe methods to effectively decellularize and demineralize porcine molar tooth buds, while preserving natural ECM protein gradients. Natural tooth ECM composition was assessed using histological and immunohistochemical (IHC) analyses of fibrillar and basement membrane proteins. Our results showed that Collagen I, Fibronectin, Collagen IV, and Laminin gradients were detected in natural tooth tissues, and retained in decellularized samples. Second harmonic generation (SHG) image analysis and 3D reconstructions were used to show that natural tooth tissue exhibited higher collagen fiber density, and less oriented and less organized collagen fibers, as compared to decellularized tooth tissue. We also found that reseeded decellularized tooth scaffolds exhibited distinctive collagen content and organization as compared to decelluarized scaffolds. Our results show that SHG allows for quantitative assessment of ECM features that are not easily characterized using traditional histological analyses. In summary, our results demonstrate the potential for natural decellularized molar tooth ECM to instruct dental cell matrix synthesis, and lay the foundation for future use of biomimetic scaffolds for dental tissue engineering applications.

Decellularized Tooth Bud Scaffolds for Tooth Regeneration

“…As use of decellularized scaffolds has grown in popularity, decellularization methods have tailored to preserve tissue-type specific ECMs (Guyette et al 2014). Previously, our laboratory published methods to generate dTB ECM scaffolds that supported in vitro cultured DE and DM cell attachment and proliferation (Traphagen et al 2012). These studies identified the gentlest approach to completely decellularize porcine TBs, while retaining nTB ECM gradients.…”

“…Porcine TB decellularization was performed as we previously described (Traphagen et al 2012). Any mineralized tooth cusps, if present, were removed after the decellularization process, prior to cell seeding.…”

A biocompatible decellularized pulp scaffold for regenerative endodontics

“…Within the dental field attempts to decellularize porcine tooth buds (Traphagen et al . , Zhang et al . ), pulp tissues obtained from miniature swine teeth (Chen et al .…”

Generation of Bioartificial Salivary Gland Using Whole-Organ Decellularized Bioscaffold

“…The decellularization of SMGs was based on published methods [Gilbert et al, 2006;Traphagen et al, 2012]. Briefly, harvested glands were placed in 10% sodium dodecyl sulfate (SDS; Sigma) in deionized water (w/v) for 32 h at 25 ° C with constant rocking in a shaker (MaxQ, Thermo Scientific, USA), and the SDS solution was changed every 8 h. The samples were then washed with enough sterile, deionized water with constant rocking in the shaker at 25 ° C for 4 h to remove residual SDS.…”