Human Freeze-dried Dentin Matrix As A Biologically Active Scaffold For Tooth Tissue Engineering

Tooth loss results from dental caries, periodontal disease, trauma, et al. Nowadays,tooth loss is replaced with dental prosthesis or dental implants, while there are still greatdifference between them and natural tooth. The traditional denture is restricted by thecondition of residual teeth and alveolar crest and could not completely restore themasticatory efficiency. Despite reported clinical success, dental implant failure is welldocumented in the literature including peri-implant bone loss and infections, which resultfrom lacking of periodontal tissues and effective epithelial cell sealing.A promising method for tooth loss is tooth regeneration by tissue engineering, whilelacking of ideal scaffold is the major limiting factor. Biological scaffolds provide anatomicdimensions of matrices in which cells synthesize tissues and, therefore, play important, ifnot indispensible, roles in tooth regeneration. Scaffolds provide biophysical support forcell recruitment, adhesion, proliferation, differentiation, and/or metabolism. For toothregeneration, the scaffold should contain rich proteins and factors for odontogenesis. Inaddition, proper biocompatibility, mechanical properties and ease of preservation andtransport are necessary for tooth regeneration. Dentin is one of the main components ofteeth and has been used as an active scaffold to accommodate odontogenic cells. However, although allogenic dentin has a low immunogenicity, its biological molecules are at risk ofbeing damaged during storage and transportation. In this study, we developed a modifiedfreeze drying technique to prepare a freeze-dried dentin matrix (FDDM) that preserves thebioactivity of dentin but minimizes the immune responses of outside cells. In this study,the mechanical properties were tested and followed with in vitro and in vivo test to studythe biocompatibility and odontogenesis of freeze dried dentin matrix (FDDM).Part I FDDM’s fabrication and mechanical properties testingObjectives(1) Fabrication of freeze dried dentin matrix scaffold.(2) Evaluation of FDDM’s mechanical properties and COL-1releasing capacity.Methods(1) Teeth were preserved in an ultra-low temperature chill3months to minimize theirimmunogenicity. Then, the periodontal ligament (PDL), outer cementum, dental pulp,predentin, and enamel were thoroughly removed. Afterwards the dentin was degreasedand freeze dried. Finally, FDDM were sterilized by Co-60irradiation, packaged,sealed, and preserved at room temperature.(2) The mechanical properties of FDDM were determined, including the compressionresistance, flexural strength, flexural modulus, and microhardness. Dentin (D) andhydroxyapatite/tricalcium phosphate (HA) samples were used as controls. Anenzyme-linked immunosorbent assay (ELISA) was used to investigate the collagentype I (COL-1) release from FDDM, D, and HA. The structures of FDDM, D, and HAwere observed using a scanning electron microscope（SEM）. For histological analysis,hematoxylin and eosin (HE) staining were used.Results(1) The fabricated FDDM was intact and has similar shape, size compared with dentin.(2) The compression resistance test showed that there was no statistically significantdifference between the mean compressive force of FDDM and D, however, the meancompressive force required to break FDDM and D was much higher and statistically different than that of HA (p<0.01)(Fig.1D). Similar trends were observed for theelastic modulus of compression, flexural strength, and flexural modulus of FDDM, D,and HA (p<0.01). Concentrations of COL-1and materials’ structure of both FDDMand D were similar.Conclusions(1) FDDM were successfully fabricated.(2) FDDM has mechanical and biological characteristics similar to those of dentin.Part II Evaluation of FDDM’s biocompatibility in vitroObjectives(1) Isolation and identification the biological characters of DPSCs(2) Test of FDDM’s biocompatibility in vitroMethods(1) DPSCs were isolate from human dental pulp. Clone formation assay was used todetect proliferarion capacity. The osteoblastic/adipogenic differentiation potential ofDPSCs were invesgated. The CD markers of DPSCs were analyzed by flow cytometryanalysis.(2) To investigate FDDM’s effect to DPSCs, biological characteristics of DPSCs includingcell morphology, cell proliferation, collagen secretion, and gene and protein expression,were investigated using various in vitro testing models.Results(1) The results showed that the cultured cells could form adherent clonogenic cell clustersand could form mineralized nodules and lipid droplets. The DPSCs were positive forSTRO-1, CD29, CD90, CD105, and CD44and negative for hematopoietic markersCD34and CD45and endothelial cell marker CD31.(2) DPSCs cultured on FDDM and dentin demonstrated superior attachment, growth,viability, and collagen secretion ability but decreased mineral capability compared toDPSCs cultured on HA (P<0.05). Conclusions(1) The isolated and cultured cells were DPSCs.(2) FDDM has superior biocompatibility and could induce DPSCs differentiated towardsodontoblast in vitro.Part III FDDM as an inductive scaffold for dentin-pulp complexregeneration in vivoObjectivesTo evaluate FDDM’s biocompatibility and odontogenesis in vivoMethodsTo assess the inductive capacity of FDDM in vivo, a combination of FDDM andhuman dental pulp stem cell (DPSC) sheets were subcutaneously implanted in the dorsalpocket of nude mice. At8weeks post-implantation, the transplants were removed andhistologically studied. HE and immunohistochemical staining were used in this part.ResultsComplete dentin-pulp complexes were regenerated by DPSC sheets combined witheither D or FDDM, and polarizing odontoblast-like cells aligned with newly secretedpredentin-like tissues. Abundant blood vesselswere observed in the pulp-like tissuesgenerated by FDDM and no inflammatory response was observed. Typical dentin-pulptissue was not regenerated in the inner holes of HA, however, poorly organized tissuecontaining a few cells and a sparsely secreted collagen matrix was observed. Moreover,obvious inflammatory cell infiltration was observed in the inner holes of the HA scaffold.Both FDDM and dentin was positive for ALP, COL-3, DSP, fibronectin, and humanmitochondriaConclusionsHistological results show that FDDM, which is similar to dentin, supporteddentin-pulp-like tissue regeneration in vivo. FDDM constitutes a novel bioinstructive scaffold for tooth tissue engineering.