**Multilineage Di** ff**erentiation Potential of Human Dental Pulp Stem Cells—Impact of 3D and Hypoxic Environment on Osteogenesis In Vitro**

**Anna Labedz-Maslowska 1,**†**, Natalia Bryniarska 1,2,**†**, Andrzej Kubiak 1,3,**†**, Tomasz Kaczmarzyk 4, Malgorzata Sekula-Stryjewska 5, Sylwia Noga 1,5, Dariusz Boruczkowski 6, Zbigniew Madeja 1 and Ewa Zuba-Surma 1,\***


Received: 28 July 2020; Accepted: 25 August 2020; Published: 26 August 2020

**Abstract:** Human dental pulp harbours unique stem cell population exhibiting mesenchymal stem/stromal cell (MSC) characteristics. This study aimed to analyse the di fferentiation potential and other essential functional and morphological features of dental pulp stem cells (DPSCs) in comparison with Wharton's jelly-derived MSCs from the umbilical cord (UC-MSCs), and to evaluate the osteogenic di fferentiation of DPSCs in 3D culture with a hypoxic microenvironment resembling the stem cell niche. Human DPSCs as well as UC-MSCs were isolated from primary human tissues and were subjected to a series of experiments. We established a multiantigenic profile of DPSCs with CD45−/CD14−/CD34−/CD29+/CD44+/CD73+/CD90+/CD105+/Stro-1+/HLA-DR− (using flow cytometry) and confirmed their tri-lineage osteogenic, chondrogenic, and adipogenic di fferentiation potential (using qRT-PCR and histochemical staining) in comparison with the UC-MSCs. The results also demonstrated the potency of DPSCs to di fferentiate into osteoblasts in vitro. Moreover, we showed that the DPSCs exhibit limited cardiomyogenic and endothelial di fferentiation potential. Decreased proliferation and metabolic activity as well as increased osteogenic di fferentiation of DPSCs in vitro, attributed to 3D cell encapsulation and low oxygen concentration, were also observed. DPSCs exhibiting elevated osteogenic potential may serve as potential candidates for a cell-based product for advanced therapy, particularly for bone repair. Novel tissue engineering approaches combining DPSCs, 3D biomaterial sca ffolds, and other stimulating chemical factors may represent innovative strategies for pro-regenerative therapies.

**Keywords:** stem cells; dental pulp stem cells; osteogenesis; biomaterials; tissue engineering; regenerative medicine
