Evaluation of in vitro biocompatibility of human pulp stem cells with allogeneic, alloplastic, and xenogeneic grafts under the influence of extracellular vesicles

Therapies using dental pulp stem cells (DPSCs) or stem cell-derived extracellular vesicles (EVs) have shown promising applications for bone tissue engineering. This in vitro experiment

evaluated the joint osteogenic capability of DPSCs and EVs on alloplastic (maxresorp), allogeneic (maxgraft), and xenogeneic (cerabone) bone grafts. We hypothesize that osteogenic

differentiation and the proliferation of human DPSCs vary between bone grafts and are favorable under the influence of EVs. DPSCs were obtained from human wisdom teeth, and EVs derived from

DPSCs were isolated from cell culture medium. DPSCs were seeded on alloplastic, allogeneic, and xenogeneic bone graft substitutes for control, and the same scaffolds were administered with

EVs in further groups. The cellular uptake of EVs into DPSC cells was assessed by confocal laser scanning microscopy. Cell vitality staining and calcein acetoxymethyl ester staining were

used to evaluate cell attachment and proliferation. Cell morphology was determined using scanning electron microscopy, and osteogenic differentiation was explored by alkaline phosphatase and

Alizarin red staining. Within the limitations of an in vitro study without pathologies, the results suggest that especially the use of xenogeneic bone graft substitutes with DPSCS and EVs

may represent a promising treatment approach for alveolar bone defects.

Bone defects of the alveolar ridge usually occur as a result of tooth loss, inflammation, trauma, or pathology and pose major problems during dental or implant rehabilitation. Augmentation

procedures thus aim to generate sufficient bone volume for implant placement1. Different bone grafting materials can be used for the augmentation of bone defects. Because of its

osteogenetic, osteoinductive, and osteoconductive properties, bone grafts of autologous origin represent the gold standard for the therapy of bony defects in the human jaw, although they are

associated with major drawbacks such as donor-side morbidity2, multiple required surgeries, and increased operation times3. In addition to autologous bone, allogeneic, xenogeneic, and

alloplastic bone graft substitutes (BGSs) can be used for the augmentation of bone defects. Bone substitutes offer the advantage of eliminating harvest morbidity, but they are currently

inferior to autologous bone in terms of osteogenesis, osteoinduction, and osteoconduction. Much research has thus been conducted to improve the properties of BGSs.

Multipotent mesenchymal stem cells (MSCs) are adult stem cells that can be isolated from a wide variety of tissues4,5,6,7. In 2000, MSCs were isolated for the first time from the healthy

pulp tissue of extracted third molars8. In addition to their rapid proliferation and colony-forming abilities9, MSCs derived from DPSCs have indicated strong differentiation potential to

osteoblasts, adipocytes, chondrocytes, odontoblasts, and neurons10. Compared to MSCs derived from bone marrow, DPSCs have a higher proliferation rate10, are clonogenic cells, and have

promising regenerative potential to treat severe tissue damage, all of which make DPSCs a frequently studied stem cell type among MSCs in research. The ability to differentiate into

bone-forming cells make DPSCs a promising therapeutic option in the regenerative medicine of bone deficiency3,11.

Recent evidence suggests that the beneficial regenerative effects of stem cell-based therapy are most likely orchestrated by their paracrine processes9,12,13,14,15,16,17. EVs are secreted by

cells in a paracrine manner, are surrounded by a lipid bilayer structure, and have a diameter of about 150–180 nm. They contain genetic substances such as microRNAs (miRNAs) and EVs are

part of cell-to-cell communication in the form of exosomes or microvesicles18,19,20,21. MicroRNAs are non-coding RNA components that inhibit the translation of mRNA. Thus, they are an

elementary component of cell proliferation and cell differentiation22. The target specificity and modulatory genes of miRNAS are related to osteogenesis, and consequently promote osteogenic

differentiation of MSCs and thus induce bone tissue regeneration23.

Impairment by degradative enzymes has often limited the use of miRNAs in bone remodeling studies in the past24. These limitations could be overcome by the production of naturally derived

EVs, since these vesicles can be used to transport miRNAs to target cells25. EVs have the advantage of being easily obtained from body or cell culture fluids26,27, and they are characterized

by stability with low susceptibility, elicit little immune response if any, and have an intrinsic homing effect, which favors the uptake of EVs with osteogenic miRNAs by target cells28. New

approaches to stem cell therapy are thus being expanded to include the use of EVs9,13.

Considering that EVs and DPSCs play a major role in cell-based therapies16, and that EVs can potentially modulate the proliferation and differentiation of DPSCs in osteogenesis, an

investigation of DPSCs on different scaffolds and EV interactions seems essential to the development of new therapies for bone defects. Currently, an insufficient number of studies have

investigated the osteogenic cell response of DPSC under the influence of EVs on different conventional BGSs in vitro. The aim of this study was thus to investigate the internalization of EVs

into DPSCs and to determine the influence on the attachment, growth, and osteogenic differentiation of DPSCs on alloplastic, xenogeneic, and allogeneic bone scaffolds under the influence of

EVs.

As previously described29, DPSCs were generated from 3 impacted third molars extracted in healthy adult males in the Oral and Maxillofacial Surgery Department of RWTH Aachen University. All

methods were carried out in accordance with relevant guidelines and regulations of the ethical approval by the Ethics Committee of the Medical Faculty of RWTH Aachen University (EK 374/19).

Written informed consent was obtained from all participants. The teeth were placed in chlorhexidine before being opened with sterile dental fissure drills at the cementoenamel junction.

Sterile barbed broaches were used to dissect pulp tissue from the crown and root. Prior to the isolation of DPSC, a mixed enzymatic solution was prepared using 1 ml of collagenase type I (12

 mg/ml), 1 ml of dispase (16 mg/ml) in 2 ml of sterile phosphate-buffered saline (PBS) containing 100 mg/ml of penicillin, and 100 mg/ml of streptomycin. The minced pulp tissue was

transferred into the prepared enzyme solution for digestion for 60 min at 37 °C and vortexed every 30 min. Using a 70 μM cell sieve, any large-cell aggregates were removed to obtain a

single-cell suspension. Digestion was terminated by the addition of 3 ml of minimal essential medium (MEM) containing 10% (v/v) fetal bovine serum (FBS). The single-cell suspensions were

then centrifuged at 1,000 rpm for 5 min at room temperature. After removal of the supernatant, the pellets were resuspended in 1 ml of cell medium and cultured in a 25 cm2 cell culture dish

at 37 °C in an incubator at 5% CO2 atmosphere and 100% relative humidity. After approximately 3–5 days, the adherent DPSCs migrated outward from the pulp-tissue pieces. Briefly, the isolated

DPSCs were cultured in EV-free basic MEM medium (growth medium) containing 10% (v/v) FBS and 1% (v/v) antibiotic–antimycotic solution at 37 °C in an incubator with 5% CO2 atmosphere and

100% relative humidity. Refreshing of the medium was performed every two days. DPSCs in stages 3 to 5 were used in the subsequent trials. Induction of osteogenic differentiation was

conducted by culturing DPSCs in osteogenic differentiation medium supplemented with 0.1 mM ascorbic acid, 100 nM dexamethasone, and 10 mM β-glycerophosphate.

After reaching roughly 80% confluence, DPSCs were rinsed twice with PBS and cultivated for another 24 h in medium without FBS. To remove cellular debris, and any unattached cells, the

supernatants were collected, differentially centrifuged (300 g, 2.000 g, and 5.000 g) for 15 min at 4 °C, and filtered with a 0.22 μm filter; the sediment was then discarded. The supernatant

was then transferred to an ultracentrifuge tube and subjected twice to ultracentrifugation at 20,000 g for 90 min at 4 °C to pellet DPSC-derived EVs. The pellets were re-suspended in 100 μL

of sterile PBS to obtain homogeneous EV suspension and then stored at − 80 °C for further application.

Nanoparticle tracking analysis (NTA) was then used to test quantitative concentration and the particle size distribution of DPSC-derived EVs. All samples were diluted to a final volume of 1 

mL using PBS. A pre-test was performed to evaluate the ideal measurement concentration of 20–100 particles per frame; see Fig. 1a. Settings were implemented according to the manufacturer’s

software manual (NanoSight NS300). In addition, the morphology and size of the isolated DPSC-EVs were assessed by transmission electron microscopy (TEM) (Fig. 1b–d). A total of 10 μL of

DPSC-EVs was pelleted, resuspended in 10 μL of HEPES buffer, and dropped onto a carbon-coated copper grid for TEM observations.

Examination of characteristics of DPSC-derived EVs: The number and sizes of EVs were analyzed by (a) nanoparticle tracking analysis (NTA) with NanoSight NS300. (b–d) The EVs shape were

examined by transmission electron analysis (TEM). e Illustrates the cellular internalization of WSA-labeled EVs (red fluorescence) into DAPI positive DPSC cells (blue fluorescence) after 1 

h, 3 h and 8 h.

Commercially available particulate bone grafts (particle size of 0.5–2 mm) were used in this in vitro study. We used maxresorb (botiss biomaterials GmbH, Germany) as a biphasic calcium

phosphate (BCP) alloplastic scaffold. For the allogeneic scaffold, we used maxgraft (botiss biomaterials GmbH, Germany), a freeze-dried bone allograft. For the xenogeneic bone graft, we used

cerabone (botiss biomaterials GmbH, Germany), a deproteinized bovine bone mineral.

First, the isolated DPSC-derived EVs were fluorescently labeled with a molecular lectin probe, Wheat Germ Agglutinin Conjugates (WGA, Alexa Fluor 488 conjugate), for 30 min at room

temperature before being terminated by centrifugation at 20,000 g for 90 min and resuspended in PBS. These WGA-labeled EVs were co-cultured with DPSC (5 × 104/confocal dish) for different

times (1 h, 3 h, and 8 h); the cells were then rinsed with PBS and fixed in 4% paraformaldehyde. The cell nucleus was then stained with 6-diamidino-2-phenylindole (DAPI) (cat. no. 2381700,

Invitrogen, USA) for another 15 min. The cellular uptake of WGA-labeled EVs by DPSCs after different incubation times was observed using confocal laser scanning microscopy (CLSM).

Additionally, 3D reconstruction images from WGA and DAPI fluorescence were automatically formed using ZEN lite software at different time points (Fig. 1e).

After culturing for 7 days, the cellular morphologies on various bone scaffolds under EV intervention at an initial density of 20,000 cells/dish were evaluated through cytoskeleton staining

and scanning electron microscopy (SEM). For the assessment of cytoskeletal organization, the cell-scaffold samples were pre-treated with EVs and afterward rinsed twice with PBS and fixed in

4% paraformaldehyde for 20 min, followed by a pre-treatment with Triton X-100 (0.1% v/v) for 5 min. The samples were then co-stained with rhodamine-phalloidin (cat. no. R415, Invitrogen,

Germany) to visualize F-actin and DAPI (cat. no. 2381700, Invitrogen, Germany) to visualize the nuclei for 45 min and 5 min, respectively. Sample visualization was conducted after 1 h, 3 h,

and 8 h via CLSM, and Z-stack images for 3D construction were obtained.

For SEM observations, the cell-scaffold constructs in various groups were gently washed twice with PBS buffer, fixed with 2.5% glutaraldehyde for 4 h, and then dehydrated with a series of

different concentrations of ethanol (30%, 50%, 70%, 90%, and 100%) twice. Next, cell/scaffold constructs were dried overnight according to an established protocol30 and then sputtered with

Au/Pd and observed using SEM (XL30 FEG; FEI, Eindhoven, the Netherlands) with an acceleration voltage of 10 kV.

Afterward, the cell-seeded BGSs were cultivated in confocal dishes at an initial density of 20,000 cells/dish for 3 or 7 days. Half the samples underwent EV stimulation (5 × 109 

particles/mL). At each predetermined time point, the medium was discarded and the cells were washed three times. Two different stainings were conducted, per the manufacturer’s instructions:

(1) live cell calcein AM staining (cat. no. PK-CA707-30002, PromoKine, Germany) and (2) DAPI staining (cat. no. 2381700, Invitrogen, USU). DPSC cells growing on the bone scaffolds were

rinsed three times and observed via CLSM to determine the proliferative effect.

A Live/Dead Cell Assay Kit (PromoKine, PromoCell GmbH, Germany) was used to detect cell viability from intracellular esterase activity and the plasma membrane integrity of DPSCs. DPSCs were

seeded on to the three different bone graft scaffolds (20,000 cells/dish) in the osteogenic medium for 1, 3, 5, and 7 days. The scaffolds were then rinsed twice with warm PBS, and the cells

were labeled with a mixed fluorescence dye containing 2 μM calcein AM and 4 μM ethidium homodimer-III (EthD-III) for 45 min, per the manufacturer’s instructions. Finally, the ratio of green

fluorescence to red fluorescence was analyzed using ImageJ software. (Free Java software was provided by the National Institutes of Health, Bethesda, MD, USA.)

Alkaline phosphatase (ALP) staining was used to evaluate the early- and late-phase osteogenic differentiation in all groups after 7 and 14 days of cultivation. In brief, the culture medium

was aspirated, and DPSCs were gently washed with PBS buffer twice. A fixing solution was added and then incubated at room temperature. After 5 min, the fixing solution was aspirated, and ALP

staining solution (cat. no. GR3358974-1, Abcam Co., UK) was added in darkness and incubated at room temperature for a further 15 min. Observation of ALP-positive (ALP+) cells was conducted

via optical microscopy and analyzed using ImageJ software.

Alizarin red staining (ARS) (cat. no. 3512879, Merck, Germany) was used to determine the mineralized nodule formation of late-phase osteogenic differentiation by calcium deposition. After

osteogenic incubation for 14 days, the cells in various groups were rinsed twice with PBS, fixed in 4% paraformaldehyde for 10 min, and then stained with 0.1% ARS solution (40 mM, pH 4.2) in

darkness for 25 min at room temperature. These mineralized precipitates were observed and photographed via an optical microscope and analyzed using ImageJ software. The mineralized nodules

appeared as a dark-red center and a light-red peripheral area.

Analyses were performed by using Prism 7.0 (GraphPad Software, Inc., San Diego, CA, USA). Before analysis, the values were tested using D’Agostino–Pearson or Shapiro–Wilk test criteria for

normal distribution and passed the Brown–Forsythe test for equal variances. A two-way ANOVA was used to analyze the parametric data of the groups with respect to two different categorical

variables: Variable one includes the different bone material and variable two the presence or absence of EVs. A post hoc Tukey’s multiple comparison test, with individual variances computed

for each comparison, was conducted. The data in this paper represent the means ± standard deviation (SD). Differences between the groups were considered significant when p