Light-controlled scaffold- and serum-free hard palatal-derived mesenchymal stem cell aggregates for bone regeneration

Abstract

Cell aggregates that mimic in vivo cell-cell interactions are promising and powerful tools for tissue engineering. This study isolated a new, easily obtained, population of mesenchymal stem cells (MSCs) from rat hard palates named hard palatal-derived mesenchymal stem cells (PMSCs). The PMSCs were positive for CD90, CD44, and CD29 and negative for CD34, CD45, and CD146. They exhibited clonogenicity, self-renewal, migration, and multipotent differentiation capacities. Furthermore, this study fabricated scaffold-free 3D aggregates using light-controlled cell sheet technology and a serum-free method. PMSC aggregates were successfully constructed with good viability. Transplantation of the PMSC aggregates and the PMSC aggregate-implant complexes significantly enhanced bone formation and implant osseointegration in vivo, respectively. This new cell resource is easy to obtain and provides an alternative strategy for tissue engineering and regenerative medicine.

Keywords: bone regeneration; cell aggregates; hard palate; light‐controlled; mesenchymal stem cells.

FIGURE 1

Wound healing processes of palatal‐derived mesenchymal stem cells (PMSCs) and adipose‐derived mesenchymal stem cells (AMSCs) harvesting. (a) A long strip of mucosa of 1.5 mm × 3 mm was removed from the hard palate. The wound showed a pink, healthy healing appearance without infection from the second day after surgery. (b) Subcutaneous adipose tissue was removed from the inguinal region and the harvesting wound was intermittently sutured. The healing time of the wound was approximately 8 days. During this period, the wound was prone to dehiscence. (c) The healing time of PMSCs harvesting was significantly shorter than that of AMSCs. (d) All the hard palatal mucosa wounds were healed without incident, while the adipose tissue harvesting sites had a high wound dehiscence rate. *: p <0.05

FIGURE 2

Isolation, culture, and characteristics of palatal‐derived mesenchymal stem cells (PMSCs). (a) Left: Schematic representation of the rat hard palate (intraoral view). The red oval indicates the location of the sampling position. Right: Histological pattern diagram of the rat hard palate. The hard palate mucosa consisted of a keratinized epithelium layer and a lamina propria layer. PMSCs located near the basement membrane between the latter two layers. (b) The technical protocol of this study. We isolated and cultured PMSCs, and then harvested PMSC sheets using light‐activated cell sheet technology. After transferring PMSC sheets into serum‐free medium, PMSC aggregates formed and were collected under light illumination. (c) Isolated PMSCs gradually migrated from the tissues in culturing medium on day 3, 5, and 8. (d) Cell colonies formed. (e) The culture time decreased between passage 0 and passage 3 and then increased. Scale bars: (c) first row: 500 μm, second row: 200 μm, (d) first figure: 1 cm, second figure: 500 μm

FIGURE 3

Identification of palatal‐derived mesenchymal stem cells (PMSCs). (a) PMSCs from passage 1 (P1) to passage 6 (P6) were spindle‐shaped. (b) Flow cytometry analysis showed that PMSCs were positive for CD90, CD44, and CD29, and negative for CD34, CD45, and CD146. (c) The staining was positive after osteogenic, adipogenic, and chondrogenic differentiation. In the meantime, the osteogenic (BMP2, ALP), adipogenic (PPARγ, AP2), and chondrogenic (SOX9, Col2a1) gene expression levels were significantly elevated. *: p <0.05. Scale bar: (a) 200 μm, (c) first and third row: 200 μm, second row: 100 μm

FIGURE 4

Comparisons of characteristics of palatal‐derived mesenchymal stem cells (PMSCs) and other cell sources. (a) Exemplary images of four kinds of cells of wound closure. Results showed that percent wound closure was significantly higher in PMSC and gingival‐derived mesenchymal stem cell (GMSC) groups compared to bone mesenchymal stem cell (BMSC) group. (b) The osteogenic gene expression level changes in four kinds of cells. PMSCs exhibited relatively positive reaction to osteogenic induction. (c) The inflammation‐related gene expression level changes after TNF‐α stimulation. Pro‐inflammatory gene expression levels were significantly elevated. *: p <0.05. Scale bar: (a) 200 μm

FIGURE 5

Characteristics of palatal‐derived mesenchymal stem cell (PMSC) sheets. (a) PMSCs proliferated on the nanodot platforms from day 1 to 5, and finally formed cell sheets. (b) PMSC sheets could heal rapidly in 24 h after injury. (c) The harvested PMSC sheets could reattach onto plates. (d) Large amounts of cells and fibronectin could be observed in the PMSC sheets. PMSC sheets were positive for CD90. (e) Few cells (0.12%) lived after immersion in PFA, while most cells in the PMSC sheets and light‐activated cell sheets survived (99.98% and 99.50%, respectively). Scale bars: (a) first three figures: 200 μm, last figure: 1 cm, (b,c) 500 μm, (d) first, second, and fourth lines: 100 and 200 μm, third line: 25 μm, (e) 200 μm

FIGURE 6

Fabrication of palatal‐derived mesenchymal stem cell (PMSC) aggregates using light‐controlled TiO₂ nanodot platform and a serum‐free method. (a) The scanning electron microscopy (SEM) image showed that nanodots were evenly distributed on the TiO₂ platforms. The energy‐dispersive X‐ray spectroscopy (EDS) results showed that O and Ti are two major elements on the TiO₂ nanodot platforms. (b) Morphology of the PMSC sheets of passage 2 (P2), passage 7 (P7), and passage 22 (P22) cultured in complete medium and serum‐free medium. (c) Evaluation of the cell growth rates of PMSCs cultured with α‐MEM containing 10% FBS and serum‐free medium. Scale bars: (a) first figure: 1 and 2 μm, second and third figures: 1 μm, (b) first and second lines: 100 μm, third and fourth lines: 200 μm

FIGURE 7

Characteristics of palatal‐derived mesenchymal stem cell (PMSC) aggregates. (a) The energy‐dispersive X‐ray spectroscopy (EDS) analysis indicated that C, O, and Ti could be detected in the cell aggregates. The scanning electron microscopy (SEM) images showed that cell aggregate was composed of cells and rich extracellular matrix (ECM). (b) Live‐dead staining of the PMSCs at each step. The cells lost viability in PFA (first line), while the live cells had good viability before illumination (second line), after light illumination (third line) and after reattachment (fourth line). (c) Cell sheets on TiO₂ nanodot platform transferred into cell aggregates after culture in serum‐free medium. When coated with FBS or placed back into complete medium, spindle‐shaped cells migrated from cell aggregates. Scale bar: (a) first row: 100 μm, second row: 200 and 10 μm, (b) 200 μm, (c) first row: 200 μm

FIGURE 8

Bone regeneration evaluation of palatal‐derived mesenchymal stem cell (PMSC) aggregates in vivo. (a) Immunohistochemistry images of tibia defect healing with blank SLA implants and the PMSC aggregates‐implant complexes after 4 and 8 weeks. The expressions of BMP2 and Runx2 were significantly elevated in the PMSC aggregate group at the two healing points. (b) Hard tissue sectioning images of tibia defect healing with blank SLA implants and the PMSC aggregates‐implant complexes after 4 and 8 weeks. Bone volume/tissue volume (BV/TV) and bone‐implant contact (BIC) were measured. 3D bone regeneration evaluation in a tibial implant model (c) and a tibial defect model. (c) showed 3D images of tibia defect healing with blank SLA implants and the PMSC aggregates‐implant complex after 4 and 8 weeks, while (d) showed images of tibia defect healing without and with PMSC aggregates after 4 and 8 weeks. Multiple‐comparison analysis of BV/TV, Tb.N, Th.Sp, and trabecular thickness (Tb.Th) were performed. The PMSC aggregates significantly promoted bone regeneration. BV/TV and Tb.N were significantly elevated, while Tb.Sp was significantly decreased in the PMSC aggregate groups. *: p <0.05. Scale bars: (a) 100 μm, (b) 250 μm, (c) 5 and 1 mm, (d) 500 μm