In recent years, physical, chemical, and enzymatic methods have been considered as the most common approaches to discern tissue decellularization. The combined use of the above methods yields the best decellularization ECM. As of our study, following the basic rules of decellularization, we developed a protocol for the generation of cellular biocompatibility decellularized matrix from the thyroid gland with similar characteristics to native ECM properties as an important material for thyroid tissue engineering.
Information on thyroid decellularization protocol is limited. Though, the first attempt was by Pan et al. , who prepared the rat decellularized thyroid ECM by perfusion through the common carotid artery. The isolating and perfusing the common carotid artery in rats was challenging, therefore, making their protocol difficult to adapt to other researchers. In our study, we exposed rabbit thyroid gland tissue to ionic detergents (SDS), as well as to mechanical agitation, and chose the most appropriate solution protocol to prepare ECM. In our previous experiment, we compared the decellularization efficiency of SDS and non-ionic detergent Triton X-100,and determined the SDS as the most effective in the decellularization of rabbit thyroid gland tissue (data not shown). Besides, studies have reported better decellularization outcomes with SDS than Triton X-100.[22, 23] Thus, in this study, SDS was used as the cell removal agent. Immersion/Agitation for 72 h in 1% SDS can effective removal of cellular nuclei and cell components in the thyroid gland tissue. Also, the effects of different agitation frequency (50 rpm, 100 rpm, and 150 rpm) on cell removal were assessed. It was found that cells could not be adequately removed at 50 rpm, thus the degree of ECM destruction was increased to 150 rpm (data not shown). We revealed that agitation frequency at 100 rpm was the optimum condition for the decellularization of the thyroid. Collectively, this protocol not only removes DNA in thyroid gland tissue but also preserve the 3D spatial structure of the native thyroid gland.
The aims for successful decellularization are to clear cellular and the retention of ECM components such as a biologic cytokine, biomechanical properties, and 3D spatial structure. However, the DTG scaffolds from our protocol achieved the stringent criteria: they lack histologically visible cellular material (DAPI or H&E staining) and with under 50 (ng / mg dry tissue) concentration of the dsDNA. Since the residual DNA fragments in decellularization ECM are directly correlated to immunological rejection response upon implantation, therefore, the attained criteria are paramount. The DAPI and H&E staining showed no visible cellular nuclei in DTG scaffolds. Besides, the DNA quantification showed significantly low concentration than the above criteria, however, the DNA removal rate was 93%. Furthermore, the decellularization efficiency in our study was consistent with previous studies with accepted standards for organ decellularization.
Notably, maximum preservation of ECM composition and cytokines are important for organ regeneration. Thus, in our study, the immunofluorescence staining demonstrated the main ECM proteins in DTG scaffolds were retained, including LN, FN, Collagen type I, and IV, which are consistent with Pan et al findings. Studies have reported that Collagens are responsible for for maintaining the ECM structure. Although it is widely considered decellularization with SDS is related to ECM ultrastructure disruption[27, 28] but our SEM results demonstrated that the 3D ECM structures were preserved thus indicating that 1% SDS was safe to DTG scaffolds. Besides, LN and FN are involved in cell adhesion. Research has shown that FN can promote cell adhesion and migration. Although the rabbit thyroid gland tissues were completely decellularized, the important cytokines (VEGF, TGF-β, HGF, and CTGF) were preserved in the DTG scaffolds. The cytokines together with FN, maybe contributing to cell growth and adhesion when the HTFCs are seeded into scaffolds.
Production of decellularized scaffolds that approximates biomechanical properties of native ECM is vital in organ bioengineering, as it contributes to maintaining the structural integrity of scaffolds after transplantation and appropriate cell-matrix interaction. Studies have demonstrated that changes in ECM biomechanical properties occur in varying degrees after decellularization. Therefore, we purposely evaluated biomechanical properties of the decellularized thyroid gland and from the ECM findings, it was evident that the elastic modulus and toughness properties showed similarity to native thyroid gland tissue. As of this, it can be attributed that elastin and collagen content of DTG scaffolds were preserved. Although the impact of DTG scaffolds mechanical properties on thyroid gland cell function has not been further elucidated in this study, it has great significance for decellularization thyroid transplantation in the future.
Thyroid structure reconstruction and endocrine function restoration are the ultimate goals in thyroid organ bioengineering and regeneration. Numerous studies have explored methods to reconstruct thyroid gland tissues.[32–34] Toda et al. were the first researchers to reconstruct the thyroid follicles in three-dimensional collagen gel (main component is acid-soluble type I collagen) in an in vitro experiment. Besides, Toni et al. rebuilt the stromal/vascular scaffolds of the human thyroid gland via in vivo visualization and computer techniques. Synthetic scaffolds can meet some biological engineering requirements, but may lack complete native ECM components and bioactive factors, which are specifically required for thyroid gland cell attachment, migration, proliferation. Therefore, decellularization scaffolds seem to provide an attractive strategy to satisfy the organ bioengineering needs. Native thyroid decellularization ECM scaffolds have the potential to offer proper microenvironment for thyroid gland cells. Despite recellularization ECM materials implemented in some organs, the development of thyroid gland bioengineering significantly later than other organs, as only pan et al reported successful recellularization of native thyroid gland ECM so far. In addressing the recellularization of the DTG scaffolds issue, in our study, two steps were adopted In the first step we explored whether DTG scaffolds displayed toxicity to HTFCs. Due to the decellularization detergent SDS used in our study, it may be toxic to host cells when the DTG scaffolds are implanted. However, the scaffolds expressed no effect to proliferative activity when HTFCs were exposed to DTG scaffolds, it is indicative the residual SDS was successfully removed or was below the safe level. On the second step, thyroid follicular cells were seeded onto DTG scaffolds to assess the ability of cell adhesion and proliferation in vitro. The HTFCs adhered to the DTG scaffolds and subsequently infiltrate deeper into the scaffolds where the cells preserved the TPO expression 3 days post-seeding. The TPO expression declined after 1 week, probably due to deficiency in essential hormones, such as thyroxin stimulating hormone. Besides, DTG scaffolds can improve proliferation which was detected by CCK-8. These results illustrate that seeded cells and DTG scaffolds can maintain well biocompatibility. Also, these characteristics contributed to thyroid gland regeneration which is crucial in organ bioengineering.
In our study, we have demonstrated the DTG scaffolds with characteristics of native thyroid, have good biocompatibility, and can promote thyroid cell proliferation with important significance in thyroid organ bioengineering and regeneration. We have explored its properties preliminary in vitro, and we will implant the scaffold cell co-culture system in vivo and examine thyroid hormone expression next.