The Four Pillars for Successful Regenerative Therapy in Endodontics: Stem Cells, Biomaterials, Growth Factors, and Their Synergistic Interactions

Table 2

In vivo and in vitro studies reported.

Authors and number of cite	Year	Study design	Stem cells	Scaffold	Bioactive molecules	Most relevant findings
Dissanayaka et al.	2014	In vivo in immunodeficient mice	Dental pulp stem cells (DPSCs) prevascularized by human umbilical vein endothelial cells (HUVECs)	No	No	(i) After four-week implantation, tooth-root slices containing microtissue spheroids resulted in well-vascularized and cellular pulp-like tissues.; (ii) Immunohistochemical staining indicated that the tissue found in the tooth-root slices was of human origin.; (iii) Vascular structures formed by HUVECs in vitro were successfully anastomosed with the host vasculature upon transplantation in vivo.
Dissanayaka et al.	2015	In vitro	Dental pulp stem cells (DPSCs) prevascularized by human umbilical vein endothelial cells (HUVECs)	Agarose micromolds	No	(i) DPC microtissue microenvironment supported HUVEC survival and capillary network formation in the absence of a scaffolding material and external angiogenic stimulation.; (ii) Immunohistochemical staining for CD31 showed the capillary network formed by HUVECs did sustain, for a prolonged period.; (iii) Induced, prevascularized macrotissues showed enhanced differentiation capacity compared with DPC alone macrotissues, as shown by higher osteo-/odontogenic gene expression levels and mineralization.
Dissanayaka et al.	2015	In vivo in severe combined immunodeficient (SCID) mice	Human umbilical vein endothelial cells (HUVECs) and/or dental pulp stem cells (DPSCs)	Peptide hydrogel PuraMatrix^TM	No	(i) DPSCs increased early vascular network formation by facilitating the migration of HUVECs and by increasing vascular endothelial growth factor (VEGF) expression.; (ii) Both the DPSC monoculture and coculture groups exhibited vascularized pulp-like tissue with patches of osteodentin after transplantation in mice.; (iii) The cocultured groups exhibited more extracellular matrix, vascularization, and mineralization than the DPSC monocultures in vivo.; (iv) The DPSCs play a critical role in initial angiogenesis, whereas coordinated efforts by the HUVECs and DPSCs are required to achieve a balance between extracellular matrix deposition and mineralization.
Li et al.	2016	In vivo with 12 immunocompromised nude mice	Dental pulp stem cells (DPSCs)	Growth factor-loaded nanofibrous microsphere scaffolding system with a nanofibrous poly(l-lactic acid) (PLLA) microsphere	Vascular endothelial growth factor (VEGF)	(i) This hierarchical microsphere system not only protects the VEGF from denaturation and degradation but also provides excellent control of its sustained release.; (ii) Nanofibrous PLLA microsphere integrates the extracellular matrix-mimicking architecture with a highly porous injectable form, efficiently accommodating dental pulp stem cells (DPSCs) and supporting their proliferation and pulp tissue formation.; (iii) Successful regeneration of pulp-like tissues fulfilled the entire apical and middle thirds and reached the coronal third of the full-length root canal.; (iv) A large number of blood vessels were regenerated throughout the canal.
Rufas et al.	2016	In vitro	Dental pulp stem cells (DPSCs)	Minimal essential medium	C3a	(i) Addition of recombinant C3a induced a significant proliferation of fibroblasts and DPSCs.; (ii) When subjected to a C3a gradient, DPSCs were mobilized but not specifically recruited, whereas pulp fibroblasts were specifically recruited following a C3a gradient.; (iii) C3a is involved in increasing DPSCs and fibroblast proliferation, in mobilizing DPSCs, and in specifically guiding fibroblast recruitment.
Wang et al.	2016	In vitro	Human dental pulp cells (hDPCs)	Polycaprolactone/submicron bioactive glass hybrid composites	Cells were cultured in sterile regular medium, supplemented with 10% fetal bovine serum,100 U/mL penicillin, and 100 mg/mL streptomycin	(i) Crystalline apatite was not precipitated on pure PCL and did not exhibit precipitation.; (ii) Surface deposition on PCL/smBG hybrids was thicker than on pure bioactive glass scaffolds at a later stage.; (iii) Human dental pulp cells had a significantly higher proliferation rate on the PCL/smBG hybrid than on the bioactive glass and PCL scaffolds.; (iv) The integration of smBG into the hybrid scaffold significantly promoted the expression of markers for odontogenic differentiation.; (v) More mineralized nodules were generated in the PCL/smBG group than in the other 2 groups.
Yadlapati et al.	2017	In vitro and in vivo with 5 female C57BL/6 mice	Human stem cells from apical papilla (SCAP) and NIH-3T3 mouse fibroblasts	A biodegradable drug-loaded fiber realized by a polydioxanone fiber 50 μm in diameter	Vascular endothelial growth factor (VEGF)	(i) Enzyme-linked immunosorbent assay verified detectable concentrations of released VEGF in solution for 25 days.; (ii) No cytotoxicity was observed on stem cells of the apical papilla (SCAP) and mouse fibroblasts treated with VEGF.; (iii) VEGF treatment also induced the expression of additional growth factors with roles in tissue and blood vessel formation and neuroprotective function.; (iv) Implantation of VF and root fragments filled with VF showed biocompatibility in vivo, promoting new blood vessels and connective tissue formation into the root canal space with negligible inflammation.
Chrepa et al.	2017	In vitro	Stem cells of the apical papilla (SCAP)	Commercially available hyaluronic acid hydrogel (Restylane)	Alpha-minimum essential medium (a-MEM) (supplemented with 10% fetal bovine serum)	(i) Cell encapsulation in either Restylane or Matrigel demonstrated reduced cell viability compared with control.; (iii) Cell viability significantly increased in the Restylane group in the course of 3 days, whereas it decreased significantly in the Matrigel group.; (iii) Restylane promoted significantly greater alkaline phosphatase activity and upregulation of dentin sialo phosphoprotein, dentin matrix acidic phosphoprotein-1, and matrix extracellular phosphoglycoprotein, compared with control.
Soares et al.	2018	In vitro and in vivo	DPCs	Highly porous NF-PLLA	Simvastatin and nanofibrous poly(l-lactic acid) scaffolds to promote the odontogenic potential of dental pulp cells in an inflammatory environment	(i) Adding simvastatin significantly represses the expression of proinflammatory mediators and also reverted the negative effects of LPS on expression of odontoblastic markers in vitro and in vivo.; (ii) DPC/NF-PLLA constructs treated with LPS/simvastatin also led to an increase in vessel-like structures, correlated with increased VEGF expression in both DPSCs and endothelial cells.; (iii) Combination of low dosage simvastatin and NF-PLLA minimizes the inflammatory reaction and increases the regenerative potential of resident stem cells.
Itoh et al.	2018	In vitro and in vivo	3D DPSC constructs	No	No external growth factors	(i) Pulp-like tissues with rich blood vessels were formed within the human root canal 6 weeks after implantation.; (ii) Histologic analyses revealed that transplanted DPSCs differentiated into odontoblast-like mineralizing cells at sites in contact with dentin.; (iii) Human CD31–positive endothelial cells were found at the center of regenerated tissue.; (iv) Self-organizing ability of 3D DPSC constructs was active within the pulpless root canal in vivo.; (v) Blood vessel–rich pulp-like tissues can be formed with DPSCs without requiring scaffolds or growth factors.
Alqahtani et al.	2018	In vitro and in vivo	Human dental pulp stem cells (HDPSC)	Dental pulp extracellular matrix (DP-ECM)	No external growth factors	(i) Decellularized ECM supports cellular infiltration together with the expression of pulp-dentin and vascular markers (DSP and CD31).