Preparation and characterization of PCL/LAP membranes
PCL and PCL/5wt%LAP (referred to as PCL/LAP) composites were fabricated by a solvent-exchange method as described in our previous study[17]. PCL and PCL/LAP nanofibrous membranes were fabricated by electrospinning. The PCL or PCL/LAP composites were dissolved in hexafluoroisopropanol (HFP) to obtain an 8 wt% solution. The electrospinning of PCL and PCL/LAP was carried out at 10 kV and a flow rate of 1 mL/h at room temperature under 30% humidity. The electrospun membranes were dried overnight in a vacuum to remove the residual solvent. The morphology of both electrospun membranes was observed by scanning electron microscopy (SEM) (Hitachi S4800 FEG, Japan). The elemental analyses was performed using energy-dispersive spectroscopy (EDS) (Hitachi S4800 FEG, Japan). The mechanical properties of the both membranes (10×50 mm2) were detected by the universal testing machine (CMT-51,China). The stress-strain curves were obtained, and the tensile strength, strain at fracture and Young’s modulus were subsequently statistically analyzed. Both the PCL and PCL/LAP membranes were sterilized by ethylene oxide for the following experiments.
PDLC culture
Ethical approval for the collection of PDLCs was attained through the School and Hospital of Stomatology, Fujian Medical University. Primary hPDLCs were harvested from healthy human PDL tissue as previously described[23] and cultured in Dulbecoo’s Modified Eagle Medium (DMEM) with 10% Fetal Bovine Serum (FBS). PDLCs within passages 2 and 6 were used in the following studies.
Cytoskeleton staining
PDLCs were seeded on PCL and PCL/LAP nanofibrous membranes for 1 day and then fixed, permeabilized, and blocked to be stained with rhodamine phalloidin (1:200; Cytoskeleton, USA) and 4’,6-diamidino-2-phenylindole (DAPI) solution (Beyotime, China). The stained cells were imaged using fluorescence microscopy (Zeiss, Germany).
Live/dead cell staining
PDLCs were seeded on PCL and PCL/LAP nanofibrous membranes for 1 day and incubated with propidium iodide (PI) for dead cells (red) and calcein AM for live cells (green) for 15 min at 37 °C. Finally, the stained PDLCs on both membranes were imaged using fluorescence microscopy. The cell viability percentage was analyzed by dividing the number of live cells by the total number of cells.
Cell proliferation
ACCK-8 assay (Dojindo, Japan) was used to evaluate PDLC proliferation. PDLCs at each time point were determined after incubation with 10% CCK8 solution, and the optical density (OD) value at 450 nm was measured by using an iMark microplate reader (Bio-Rad, USA).
ALPL and mineral nodule formation assays
Alkaline Phosphatase (ALPL) staining was performed using BCIP/NBT reagent (Beyotime, China) following osteogenic induction for 7 days. For ALPL activity, PDLCs on both nanofibrous membranes were lysed with RIPA lysis buffer (Beyotime, China) and assessed using an ALPL assay kit (Jiancheng Inc., China) and a BCA protein assay kit (Beyotime, China). ALPL activity was normalized to the total protein amount in the cell lysate. For mineral nodule formation analysis, PDLCs were cultured on both membranes following osteogenic induction for 14 days, stained with 40 mM Alizarin Red S (pH 4.2) and quantified by eluting with 10% cetylpyridinium chloride in 10 mM sodium phosphate (pH 7.0).
Quantitative real-time polymerase chain reaction (PCR)
RNA was extracted from PDLCs using TRIzol reagent (Life Technologies, USA) and transcribed using a PrimeScript RT reagent kit (Takara, Japan). Gene expression was evaluated using a LightCycler 480 real-time PCR system (Roche Diagnostics, Germany) with SYBR green PCR mix (Takara, Japan). The primers (Table 1) were synthesized by Sangon Biotech (Shanghai, China).
Table 1 Primer sequences used in quantitative real-time PCR
Gene
|
Primers
|
GAPDH (homo)
|
Forward: 5′-ACCCACTCCTCCACCTTTGAC-3′
Reverse: 5′-TCCACCACCCTGTTGCTGTAG-3′
|
RUNX2 (homo)
|
Forward: 5′-ACCAGCAGCACTCCATATCTCTAC-3′
Reverse: 5′-CTTCCATCAGCGTCAACACCATC-3′
|
ALPL (homo)
|
Forward: 5′-ACTGGTACTCAGACAACGAGAT-3′
Reverse: 5′-ACGTCAATGTCCCTGATGTTATG-3′
|
COL1A1 (homo)
|
Forward: 5′-TCGGAGGAGAGTCAGGAAGG-3′
Reverse: 5′-TCAGCAACACAGTTACACAAGG-3′
|
GAPDH (mus)
|
Forward: 5’-AGGTGGTGAAGCAGGCATC -3’
Reverse: 5’-AAGGTGGAAGAGTGGGAGTTG -3’
|
IL-1β (mus)
|
Forward: 5’-CCAAGCAATACCCAAAGAAGAAGATG-3’
Reverse: 5’-TTATGTCCTGACCACTGTTGTTTCC-3’
|
IL-6 (mus)
|
Forward: 5’-GAAACCGCTATGAAGTTCCTCTCT-3’
Reverse: 5’-GTATCCTCTGTGAAGTCTCCTCTCC-3’
|
IL-10 (mus)
|
Forward: 5’-CCCCAGCCGCTTCATCCC-3’
Reverse: 5’-ACAAACAATACACCATTCCCAGAGG-3’
|
TNF-α (mus)
|
Forward: 5’-AAGGGAGAGTGGTCAGGTTGC-3’
Reverse: 5’-TGGAAAGGTCTGAAGGTAGGAAGG-3’
|
iNOS (mus)
|
Forward: 5’-GCACCACCCTCCTCGTTCAG-3’
Reverse: 5’-CCACAACTCGCTCCAAGATTCC-3’
|
ARG1 (mus)
|
Forward: 5’- GCCTTTGTTGATGTCCCTAATG-3’
Reverse: 5’- GCACCACACTGACTCTTCC-3’
|
Western blot (WB)
After 7 days of osteogenic induction, proteins of PDLCs on both membranes were extracted using RIPA lysis buffer. Proteins were fractionated by gel electrophoresis and transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, USA). The PVDF membranes were blocked and incubated with the primary antibodies RUNX2 (1:1000), ALPL (1:1000), COL1A1 (1:1000) and GAPDH (1:1000) (Abcam, USA). Then, the PVDF membranes were incubated with a secondary antibody (1:2000, Boster, China). The protein expression levels were detected using enhanced chemiluminescence reagents (Beyotime, China) and analyzed using ImageJ software.
Neutrophils treated with PDLCs-conditioned medium
PDLCs were cocultured with both nanofibrous membranes with 1 mL culture medium in 24-well plates for 7 days. The supernatants were collected, centrifuged and filtered through 0.22-µm filters. The filtrate was mixed with DMEM containing 10% FBS to obtain conditioned medium (referred to as PDLCs-PCL-CM and PDLCs-PCL/LAP-CM). The supernatants of PDLCs without membranes were used as controls (PDLCs-CM). The HL-60 cells were incubated with 1.25% DMSO to induce neutrophils and then cultured in the above conditioned medium. The neutrophil shapes were imaged using inverted microscopy. The expression levels of inflammatory genes of the neutrophils were assessed by using quantitative real-time PCR (as described in 2.7) on Day 1.
Macrophages treated with PDLCs-conditioned medium
The condition medium was obtained as described in 2.9. The RAW 264.7 macrophages were cultured in the above conditioned medium. The macrophage shapes were imaged using inverted microscopy. The expression levels of inflammatory genes of the macrophages were assessed by using quantitative real-time PCR (as described in 2.7) on Day 3. The supernatants of macrophages treated with conditioned medium were collected after 3 days of culturing. The levels of IL-1β, IL-6, IL-10 and TNF-α in the supernatants were assessed using enzyme-linked immunosorbent assay (ELISA) (Abcam, USA).
Immunofluorescent staining
Marcophages were fixed, permeabilized and blocked at Day 3. The primary antibody against iNOS (1:1000) or ARG1 (1:1000) (Abcam, USA) was incubated onto the cells for 2 h. Alexa Flour-488 conjugated secondary antibody (1:400, Abcam, USA) was added. Then, the actin cytoskeleton was stained with rhodamine phalloidin (1:200), and nuclei were stained with DAPI. The stained cells were imaged using fluorescence microscopy.
Rat calvarial defect surgical procedure
The study protocol was approved by the Animal Care and Use Committee of Fujian Medical University. All rats were anesthetized via intraperitoneal injection of 40 mg/kg ketamine. The calvarial defect model was prepared in 4-week-old rat calvarial. A round bur was used to prepare a critical-sized calvarial defect with a 6 mm diameter. The rats were split into three groups: the PCL group with PCL nanofibrous membrane placement, the PCL/LAP group with PCL/LAP nanofibrous membrane placement and the blank group without any implant as a negative control. Eight weeks later, all rats were sacrificed to collect calvarials.
Rat periodontal defect surgical procedure
The study protocol was approved by the Animal Care and Use Committee of Fujian Medical University. All rats were anesthetized via intraperitoneal injection of 40 mg/kg ketamine. The periodontal defect model was prepared in 4-week-old rat mandibles as described in a previous study[24]. A round bur was used to prepare periodontal defects with 2 mm height × 3 mm width × 1 mm depth around the first molar. The periodontium was removed to expose the root surface. The rats were split into three groups: the PCL group with PCL nanofibrous membrane placement, the PCL/LAP group with PCL/LAP nanofibrous membrane placement and the blank group without any implant as a negative control. Four weeks later, all rats were sacrificed to collect mandibles.
Microcomputed tomography (micro-CT) analysis
The fixed calvarias or mandibles samples were scanned by a micro-CT scanner (SCANCO µCT50, Switzerland). Three-dimensional images were obtained and analyzed using Mimics software (Mimics 17.0, Materialise, Leuven, Belgium). Bone volume per total volume ratio (BV/TV) and bone mineral density (BMD) were evaluated.
Histologic staining and analysis
The calvarias and mandibles were decalcified in 10% ethylenediaminetetraacetic acid (EDTA), dehydrated and embedded. Consecutive sections were obtained from the defect area and stained using hematoxylin and eosin (H&E) and Masson’s trichrome staining. The new bone areas and new attachment formation rate were assessed using ImageJ software.
Statistical analysis
Data are presented as the mean ± standard deviation. The t test or one-factor analysis of variance followed by Tukey’s HSD post hoc test was used to evaluate the differences among groups. Significance was accepted at P < 0.05 for all tests.