Dental Stem Cell-Derived Secretome/Conditioned Medium: The Future for Regenerative Therapeutic Applications

Regenerative medicine literature has proposed mesenchymal stem/progenitor cell- (MSC-) mediated therapeutic approaches for their great potential in managing various diseases and tissue defects. Dental MSCs represent promising alternatives to nondental MSCs, owing to their ease of harvesting with minimally invasive procedures. Their mechanism of action has been attributed to their cell-to-cell contacts as well as to the paracrine effect of their secreted factors, namely, secretome. In this context, dental MSC-derived secretome/conditioned medium could represent a unique cell-free regenerative and therapeutic approach, with fascinating advantages over parent cells. This article reviews the application of different populations of dental MSC secretome/conditioned medium in in vitro and in vivo animal models, highlights their significant implementation in treating different tissue' diseases, and clarifies the significant bioactive molecules involved in their regenerative potential. The analysis of these recent studies clearly indicate that dental MSCs' secretome/conditioned medium could be effective in treating neural injuries, for dental tissue regeneration, in repairing bone defects, and in managing cardiovascular diseases, diabetes mellitus, hepatic regeneration, and skin injuries, through regulating anti-inflammatory, antiapoptotic, angiogenic, osteogenic, and neurogenic mediators.

Neurite extension was promoted through the inhibition of multiple axon growth inhibitors (AGI). Therefore, SHED-CM and DPSCs-CM can be beneficial in neural regeneration.

Inoue et al., 2013 [69]
SHED-CM Cerebral stroke In vivo stroke model in rats subjected to permanent middle cerebral artery occlusion (pMCAO).
Intranasal administration of SHED-CM promoted the migration and differentiation of endogenous neuronal progenitor cells, induced vasculogenesis and improved ischemic brain injury after pMCAO.

SHED-CM Acute myocardial infarction
In vivo mouse model of ischemia-reperfusion (I/R).
In vitro Cardiac myocytes culture.
Intravenous injection of SHED-CM reduced the size of myocardial infarct, myocyte apoptosis and inflammatory cytokine levels, such as TNF-α, IL-6 and IL-β, in the myocardium following I/R. In vivo anti-collagen type II antibody-induced arthritis (CAIA), a mouse model of RA.
Single IV administration of SHED-CM provided therapeutic factors for treating CAIA including ED-Siglec-9-dependent induction of M2 macrophage polarization, inhibition of osteoclastogenesis and bone destruction.
A single injection of SHED-CM or ED-Siglec-9 significantly improved experimental autoimmune encephalomyelitis (EAE) by improving disease scores, reduced demyelination and axonal injury, reduced inflammatory cell infiltration and proinflammatory cytokine expression in the spinal cord, through shifting in the microglia/macrophage phenotype from M1 to M2.

SHED-CM Peripheral nerves
In vitro rat model of facial nerve transection.
MCP-1/sSiglec-9 in SHED-CM mediated neurological regeneration through inducing polarization of tissue-repairing macrophages, Schwann-cell bridging instead of scar formation, axonal regeneration and restoration of nerve function. Those finding suggests the potential role of SHED-CM in the regenerative treatment of severely injured peripheral nerves.
Local injection of SHED-EXs improved rat motor functional recovery and reduced cortical lesion. SHED-EXs reduced neuroinflammation by shifting microglia M1/M2 polarization.

Matsushita et al., 2017 [80]
SHED-CM Acute liver failure (ALF) In vivo d-galactosamineinduced rat model of acute liver failure (ALF) A single IV administration SHED-CM noticeably enhanced the condition of the injured liver and the animals' survival rate. SHED-CM generated an antiinflammatory/tissue-regenerating environment, together with the induction of anti-inflammatory M2-like hepatic macrophages.

SHED-CM
Spinal cord injury (SCI) In vivo rat spinal cord injury model.

SHED-CM Dental pulp regeneration
In vivo rodent orthotopic model of dental pulp regeneration in rats.
In vitro on human umbilical vein endothelial cells (HUVEC) culture.
SHED-CM showed the formation of connective tissue similar to the dental pulp inside the root canal and promoted angiogenesis.
SHED-CM promoted angiogenesis, reduced apoptosis while increased migration and vascular-like structures formation due to the presence of VEGF in SHED-CM.
In vitro SHED and hair follicle stem cells (HFSCs) culture.
Three sub-cutaneous injections of SHED-CM resulted in significantly faster stimulation of hair growth.
SHED-CM resulted in a significantly higher number of anagen-staged hair follicles and a significantly lower number of telogen-staged hair follicles.

Iohara et al., 2008 [108]
DPSCs-CM Angiogenesis In vitro culture of HUVECs DPSCs-CM provided mitogenic and antiapoptotic activities on HUVECs similar to the effect of MMP3, VEGF-A and G-CSF.
Antibody array showed the existence of a wide array of pro-and anti-angiogenic factors; including VEGF, MCP-1, PAI-1 and endostatin in DPSC-CM. DPSCs-CM significantly induced HMEC-1 migration and blood vessels formation in CAM assay.

Ishizaka et al., 2013 [89]
DPSCs-CM In vitro culture of NIH3T3 cells and human neuroblastoma cell line TGW DPSCs-CM triggered a potent anti-apoptotic activity of NIH3T3 cells and neurite outgrowth of human neuroblastoma cell line TGW.
In vitro model of retinal ganglion cells damage in rats. Early administration of DPSC-CM, particularly IGF-1, resulted in the improvement of microcirculation, tissue oxygenation, and neuroinflammation in the aSAH-injured brain.
In vitro HUVEC culture.
Single intramuscular injection of DPSC-CM into the hindlimb improved diabetic polyneuropathy, through enhancing sciatic nerve motor/sensory conduction velocity and blood flow as well as increasing intraepidermal nerve fiber density in the footpads of diabetic rats.
DPSC-CM significantly increased the in vitro proliferation of HUVECs.
Intraperitoneal injection of DPSC-CM significantly improved early neuromuscular junction (NMJ) innervation and increased motor neuron and NMJ preservation during late pre-symptomatic stages. Also, it significantly increased post-onset days of survival and overall lifespan.

GMSCs-EXs
Skin repair In vivo diabetic rat skin defect model.
Incorporation of GMSC-derived exosomes to chitosan/silk hydrogel supported healing of skin defect in diabetic rats via re-epithelialization, collagen deposition and remodeling and enhanced angiogenesis and neuronal ingrowth.
In vitro mechanically injured murine motor-neuron-like NSC-34 cells culture.
In vitro hGMSCs culture 3D-PLA scaffold enriched with hGMSCs and CM could improve osteogenesis in vitro as evident by mineralization and upregulation of osteogenesisrelated genes. In addition to in vivo induction of new bone formation and osseointegration.
In vitro hGMSCs culture.
In vitro rat Schwann cell line RT4-D6P2T culture.
GMSC-EVs promoted axonal regeneration and functional recovery of injured mice sciatic nerves. GMSC-EVs promoted in vitro proliferation, migration and upregulation of dedifferentiation/repair genes (c-JUN, Notch1, GFAP and SOX2) of Schwann cells.

GMSCs-EXs
Peripheral (Sciatic) nerve injury Injured sciatic nerve in the right hind limb in rats In vitro co-culture with Schwann cells and Dorsal Root Ganglion (DRG) cells culture Chitin conduit combined with GMSC-EXs significantly enhanced the number and diameter of nerve fibers and promoted myelin formation. Also, recovery of muscle function, nerve conduction function and motor function were observed.
GMSC-EXs significantly enhanced Schwann cell proliferation and DRG cells axon growth.

GMSCs-EXs
Tongue In vivo critical-sized tongue defect model in rats.
Small intestinal submucosa extracellular matrix with GMSCs or -GMSC-Exos constructs promoted tongue lingual papillae recovery and taste bud regeneration and reinnervation.