title: In vitro analysis of scaffold-free prevascularized microtissue spheroids containing human dental pulp cells and endothelial cells.
abstract: BACKGROUND
  Scaffolds often fail to mimic essential functions of the physiologic extracellular matrix (ECM) that regulates cell-cell communication in tissue microenvironments. The development of scaffold-free microtissues containing stem cell-derived ECM may serve as a successful alternative to the use of artificial scaffolds. The current study aimed to fabricate 3-dimensional microtissue spheroids of dental pulp cells (DPCs) prevascularized by human umbilical vein endothelial cells (HUVECs) and to characterize these scaffold-free spheroids for the in vitro formation of pulplike tissue constructs.
  
  
  METHODS
  Three-dimensional microtissue spheroids of DPC alone and DPC-HUVEC co-cultures were fabricated using agarose micro-molds. Cellular organization within the spheroids and cell viability (live/dead assay) were assessed at days 1, 7, and 14. Microtissue spheroids were allowed to self-assemble into macrotissues, induced for odontogenic differentiation (21 days), and examined for expression levels of osteo/odontogenic markers: alkaline phosphatase, bone sialoprotein and RUNX2 (Real-time PCR), mineralization (von-Kossa), and prevascularisation (immunohistochemistry for CD31).
  
  
  RESULTS
  The DPC microtissue microenvironment supported HUVEC survival and capillary network formation in the absence of a scaffolding material and external angiogenic stimulation. Immunohistochemical staining for CD31 showed the capillary network formed by HUVECs did sustain-for a prolonged period-even after the microtissues transformed into a macrotissue. Induced, prevascularized macrotissues showed enhanced differentiation capacity compared with DPC alone macrotissues, as shown by higher osteo/odontogenic gene expression levels and mineralization.
  
  
  CONCLUSIONS
  These findings provide insight into the complex intercellular cross talk occurring between DPCs and HUVECs in the context of angiogenesis and pulp regeneration and highlight the significance of developing a favorable 3-dimensional microenvironment that can, in turn, contribute toward successful pulp regeneration strategies.
authors: Department of Endodontics, Comprehensive Dental Care, The University of Hong Kong, Hong Kong SAR, China.
authors: Waruna Lakmal
authors: WL
authors: Dissanayaka
authors: Department of Endodontics, Comprehensive Dental Care, The University of Hong Kong, Hong Kong SAR, China.
authors: Lifang
authors: L
authors: Zhu
authors: Department of Endodontics, University of Texas Health Science Center at San Antonio, San Antonio, Texas.
authors: Kenneth M
authors: KM
authors: Hargreaves
authors: Periodontology and Public Health, Faculty of Dentistry, The University of Hong Kong, Hong Kong SAR, China.
authors: Lijian
authors: L
authors: Jin
authors: Department of Endodontics, Comprehensive Dental Care, The University of Hong Kong, Hong Kong SAR, China. Electronic address: zhangcf@hku.hk.
authors: Chengfei
authors: C
authors: Zhang
mesh_terms: D002450:Cell Communication; D002478:Cells, Cultured; D018920:Coculture Techniques; D003782:Dental Pulp / Q000166:cytology*; D042783:Endothelial Cells / Q000502:physiology*; D006801:Humans; D018874:Spheroids, Cellular / Q000502:physiology*; D023822:Tissue Engineering / Q000379:methods*; D014471:Umbilical Veins
keywords: Angiogenesis; prevascularization; regeneration; scaffold; tissue engineering