Keywords
periodontal cell sheet - chitosan - arginine-glycyl-aspartic acid - horizontal periodontal
defect - periodontal tissue regeneration
Introduction
Periodontal defects are frequently found in severe periodon-titis patients and various
attempts have already been performed to regenerate the new periodontal tissues.[1]
[2]
[3] The current treatments provide predictable clinical outcome of the improvement in
clinical parameters; however, these approaches are restricted in appropriate case
selection of small-to-medium-size defect. In critical size defect such as in one-wall
or horizontal periodontal defect, periodontal tissue regeneration is still a challenge
and it often results in unpredictable clinical outcome. As such focus has shifted
to the potential use of tissue engineering technique for periodontal regeneration
in large bone defect cases.[4]
[5]
[6]
[7]
[8]
[9]
The application of cell sheet for periodontal regenerative therapy has been recently
reported.[6]
[7]
[8]
[9] Thermoresponsive surfaces such as poly(N-isopropylacrylamide/ PIPAAm) give the possibility
for nonenzymatic treatment to harvest the cells, thereby protecting the cell junction,
cell surface proteins and extracellular matrix (ECM) proteins. Cells from various
sources such as periodontal ligament (PDL), bone marrow, and adipose tissues have
been tested for the synthesis of cell sheet materials.[6]
[7]
[8]
[9] Although the initial concept of cell sheet technology is to eliminate the need for
scaffold materials, due to delicate nature of the cell sheet, the use of biodegradable
support matrices is still necessary to support the cell sheet to be transplanted to
the defect areas such as periodontal tissue defects.
One of the promising biomaterials to be used for tissue regeneration is the naturally
occurring polymer of chitosan.[10]
[11]
[12]
[13]
[14] Chitosan is a partially deacetylated form of chitin, a polysaccharide present in
the exoskeleton of crustaceans shells. Its characteristics include biocompatible,
biodegradable, bioactive, and versatility in surface chemistry; all of these features
make chitosan an attractive scaffold material for tissue engineering purposes. We
previously showed that in addition to chitosan’s role as a three-dimensional scaffold
for osteogenic cells, chitosan has the capacity to stimulate dental pulp stromal cells
(DPSCs) proliferation and early osteogenic differentiation in vitro comparable to the well-known osteogenic supplement of dexamethasone.[14] Tripeptide arginine-glycine-aspartic acid (RGD) motif presents in various adhesive
proteins in the ECM and is a well-known general cell recognition motif via the cell
surface integrin receptors. The incorporation of RGD peptide to the scaffold biomaterials
was reported to improve the cell attachment to the biomaterials.[15]
[16]
Despite several studies demonstrated chitosan biomaterials in combination with RGD
peptide as a promising scaffold material for bone and cartilage tissue engineering,[17]
[18]
[19]
[20] no study reported the potential of RGD-modified chitosan to induce periodontal regeneration
in horizontal periodontal defect cases. It is currently unknown whether the combination
of periodontal cell sheet and RGD-modified chitosan could improve the formation of
periodontal tissue particularly in horizontal periodontal defect case. We hypothesized
that the addition of RGD in the chitosan scaffold could improve the periodontal tissue
regeneration capacity of PDL cell sheet. The aim of this study was to examine the
effect of PDL cell sheet and RGD-modified chitosan construct in stimulating periodontal
tissue regeneration of horizontal periodontal defect in Macaque nemestrina model.
Materials and Methods
Materials
α- Minimum Essential Medium (MEM) fetal bovine serum, penicillin, streptomycin, fungizone,
and collagenase I were all from Gibco (Life Technologies; Grand Islands, NY, USA).
RGD peptide, ascorbic acid, β-glycerophosphate, dexamethasone, and 3–[4,5- dimethylthiazol-2yl]–2,5-diphenyl-2H-tetrazolium
bromide (MTT) powder were purchased from Sigma (St. Louis, MO, United States). Dispase
was from Roche (Indianapolis, United States). All culture plates were and transwell
polyethylene terephthalate (PET) membrane were form Costar (Corning, New York, United
States). Tubes were from BD Falcon (New Jersey, United States). UpCell dish were from
Nunc; ThermoFisher Scientific, United States. Bradford Protein Assay was from Bio-Rad
protein assay kit (Bio-Rad, United States). Human cementum protein 1 (CEMP-1) enzyme-linked
immunosorbent assay kit was from Cusabio (Wuhan, China).
RGD-Modified Chitosan Preparation
Chitosan-based materials were prepared at the Center for Application of Isotope and
Radiation Technology, Indonesia National Atomic Energy Agency.[14] Chitosan with the degree of deacetylation of 94.5% was dissolved in 1% v/v acetic
acid (0.1 M) and stirred until fully dissolved to obtain a homogenous 2 wt% chitosan
solution. RGD-modified chitosan scaffold was prepared by physical adsorption of RGD
peptide to chitosan. Four milligrams of RGD peptide were added to 50 mL chitosan solution,
casted to a custom-made mold, and freezed for 24 hours. The RGD-modified scaffold
was then solidified in 1M NaOH/ethanol solution, neutralized in distilled water and
freeze-dried. Scaffold porosity was ~175 μm.[21]
Cell Culture
This protocol received an ethical clearance from Primate Research Center, Bogor Institute
of Agriculture’s Animal Care and Use Committee (ACUC No IPB PRC-15-B0012) and followed
the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines. PDLs were
collected from four Macaque nemestrina upper first incisors. Cell isolation procedures were as previously described.[14]
[22] Briefly, PDL tissue was obtained from apical two-thirds of the root. Enzymatic dissociation
was performed with 4 mg/mL collagenase I and 3 mg/mL dispase in a 15 mL tube at 37°C
under 5% CO2 in an incubator for 1 hour. Cells were then plated into six-well plate and cultured
with basal medium consisted of α-MEM, 10% Fetal Bovine Serum (FBS), 100 U/mL penicillin,
100 μg/mL streptomycin, and 1.25 μg/mL fungizone until they reached ~90% confluency
for ~14 days. Total heterogenous PDL cell population from passage 1 to 4 was used
for experiments. Cell cytotoxicity analysis was performed at 24 hours following incubation
of PDL cells and chitosan-based scaffold with MTT assay as previously described.[14] The ability of chitosan scaffold to induce cell migration was tested using 8.0 μM
Transwell PET membrane. 5.10[5] cells were seeded in the upper compartment of the well insert, while the chitosan
scaffold was placed in the lower compartment and incubated for 4 hours. The migrated
cells in the lower compartment were counted in the microplate reader (Benchmark, Bio-Rad)
at 655 nm. Data were corrected for blank values (medium only). The experiments were
repeated twice and were performed in triplicate. PDL cell sheets were obtained cultured
in 10 mm UpCell dish for 3 weeks in osteogenic medium containing basal medium supplemented
with 100 μg ascorbic acid, and 10 mM β-glycerophosphate and 10 nM dexamethasone. Prior
to transplantation, PDL cell sheets were harvested, loaded onto the chitosan-based
membrane and the cell-scaffold constructs were incubated in 37°C to facilitate cell
attachment.
Surgical Procedures
Four adults (6–8 years old) male Macaque nemestrina (weight 15–18 kg) were considered in this study. All procedures were performed under
general anesthesia using isopropyl IV (12 mg/kg body mass index) and local anesthesia
(Xylocaine-adrenalin 5 mg/mL). Horizontal periodontal defect was initially created
on four maxillary and mandibular lateral incisors using orthodontic elastic bands
in the sulcus areas.[23] However, the concavity of Macaque nemestrina tooth morphology prevented the elastic bands to stay in place; the surgical approach
was later introduced using 1.5 mm rounded-end burr to remove the surrounding alveolar
bone in 16 lateral incisors. Horizontal periodontal defect defined as a 5 × 3 mm (height
x width) of horizontal bone loss with reference point of cement–enamel junction.[24]
[25] All animals were tolerated well with the procedures. Periodontal therapy was introduced
6 weeks after periodontal defect creation that consisted of plaque control and topical
irrigation with 0.2% chlorhexidine digluconate and minocycline HCl solution. Two weeks
following periodontal therapy, lateral incisor sites were divided into four groups
based on the regenerative materials: (1) chitosan as control group, (2) RGD-modified
chitosan, (3) PDL cell sheet seeded in chitosan membrane group, and (4) PDL cell sheet
seeded in RGD-modified chitosan membrane group. Regenerative materials were securely
placed on root surfaces and their respective alveolar bone, and the flap was then
secured with 5–0 nylon sutures. Clinical periodontal parameters measurement included
pocket depth, clinical attachment loss (CAL), and bleeding on probing. An increase
in epithelial attachment was determined by subtracting CAL at 4 weeks from CAL before
cell-scaffold constructs transplantation. Gingival crevicular fluids (GCFs) were collected
every week up to 4 weeks. Biopsies consisted of lateral incisor teeth and surrounding
alveolar bone were taken 4 weeks after transplantation of the regenerative materials.
The sites were then filled with the carbonite apatite graft (GAMA-CHA; Jogjakarta,
Indonesia). This was a survival study, whereby all animals remained in the facility
following biopsy retrieval. In 6-month follow-up, all animals behaved and grew well.
Research timeline was described in [Fig. 1]. Following micro-computed tomography (micro-CT) examination, the biopsies were embedded
in paraffin, sectioned with 5 µM thickness and stained with hematoxylin and eosin
for histological analysis.
Fig. 1 Research timeline. GCF, gingival crevicular fluid; PDL, periodontal ligament.
Radiographic Examination
Digital parallel periapical radiograph was performed to measure the bone height using
cone indicator and bite registration. X-ray unit (Rextar X, 70 kV/2 mA) was used with
a 0.12 second exposure time for the anterior teeth before transplantation and each
week up to 4 weeks following transplantation. For the comparison purpose, the contrast
was adjusted on both radiographic images before and 4 weeks after treatment, using
digital subtraction radiography program, from MatLab image registration software.
Both radiograph images were overlapped and registered to obtain any density differences.
The region of interest (ROI) was 30 × 30 pixels at the top of alveolar bone whereby
the regenerative materials were transplanted. The program was then processed the average
value of the gray scale within the ROI.
CEMP-1 Protein Expression
GCFs were collected every week up to 4 weeks prior to biopsies retrieval. GCF total
protein was measured with Bradford Protein Assay and subsequently standardized up
to 200 µg/mL. CEMP-1 protein expression was measured according to manufacturer’s suggested
protocols (Cusabio).
Micro-CT Examination
Biopsies consisted of lateral incisor teeth and surrounding alveolar bone was scanned
using SkyScan 1173 (Bruker-Micro-CT; Kontich, Belgium) at a voltage of 55 kV, a current
of 145 μA, an integration time of 600 milliseconds, a resolution of 12.11 μm, and
a rotation step of 0.2 degrees. A series of projection images in a 16-bit TIFF format
was obtained from the scanning process and further reconstructed using NRecon 1.7.3.1
(Bruker-Micro-CT) using the GPUReconServer. The scanning was followed by a reconstruction
using NRecon 1.7.3.1 (Bruker-Micro-CT) with the GPUReconServer. Basic image processing
and qualitative and quantitative analyses were done using DataViewer, CTAn (Bruker-Micro-CT)
and ImageJ 1.45r (National Institute of Health; Bethesda, Maryland, United States).
Statistical Analysis
Data was statistically analyzed using GraphPad Prism 6 for MacOS X. Normality was
tested using the Shapiro–Wilk normality test. Data were analyzed using the Kruskal–Wallis
test, and significance was accepted when p < 0.05.
Results
We first tested the biomaterials in vitro. MTT assay showed biocompatibility of chitosan and RGD-modified chitosan ([Fig. 2]). Higher cell proliferation was observed in both biomaterials tested (p < 0.0001). More than 50% of the seeded cells were migrated toward lower compartments
in the Boyden chamber in both chitosan and RGD-modified chitosan group (p < 0.01) ([Fig. 3]). The initial alveolar bone height before the in vivo experiments was presented in [Figs. 4A] and served as baseline. Horizontal periodontal defect was successfully created in
the lateral incisors with the average loss of attachment of 5.45 ± 0.33 mm ([Fig. 4B]
[4C]). No significant different in the loss of attachment of lateral incisors was found
in all macaques. Four weeks following PDL cell sheet and chitosan-based scaffold construct
transplantation to the periodontal horizontal defect, an increase in epithelial attachment
was measured ([Table 1]). The clinical attachment level gain was observed in all group tested. In the group
treated with PDL cell sheet with RGD-modified chitosan, 3 mm increase in epithelial
attachment was observed. The increase was 1.7-fold higher than the group treated with
chitosan. The epithelial attachment gain reached 55% of the initial attachment prior
to horizontal periodontal defect formation as measured clinically. Alveolar bone density
was evaluated at week 4 and was shown in [Table 2]
[Fig. 4D]. Histological analysis showed the regenerated periodontal tissues in the defect
areas, while the most coronal part was still filled with fibrous tissue ([Fig. 5A]). The regenerated alveolar bone indicated by the presence of young osteocytes, characterized
by their round shaped and abundant cytoplasm due to higher metabolic activity ([Fig. 5B]). The existing bone was seen in a more apical part of alveolar bone indicated with
the presence of mature osteocytes with a more flattened shaped and with cement lines
marking the newly deposited bone ([Fig. 5B]). CEMP-1 protein expression consistently increased over period of time in all group
tested ([Fig. 6]). The highest CEMP-1 expression was observed in the group treated with PDL cell
sheet and RGD-modified chitosan. In line with this finding, the shortest distance
of alveolar bone crest and cement–enamel junction was observed in PDL cell sheet and
RGD-modified chitosan ([Figs. 7]
[8]).
Table 1
The average increase in clinical epithelial attachment
|
Group
|
Increase in epithelial attachment (mm) (Mean ± SD)
|
|
Abbreviations: PDL, periodontal ligament; RGD, arginine-glycyl-aspartic acid; SD,
standard deviation.
|
|
Chitosan
|
1.75 ± 0.71
|
|
RGD-modified chitosan
|
2.13 ± 0.83
|
|
PDL cell sheet chitosan
|
2.25 ± 0.71
|
|
PDL cell sheet RGD-modified chitosan
|
3.00 ± 0.75
|
Table 2
The average gray scale on alveolar bone density subtraction results
|
Group
|
Gray scale of alveolar bone density (Mean ± SD)
|
|
Abbreviations: PDL, periodontal ligament; RGD, arginine-glycyl-aspartic acid; SD,
standard deviation.
|
|
Chitosan
|
7.31 ± 10.27
|
|
RGD-modified chitosan
|
16.70 ± 13.17
|
|
PDL cell sheet chitosan
|
19.34 ± 21.46
|
|
PDL cell sheet RGD-modified chitosan
|
21.98 ± 7.85
|
Fig. 2 Cytotoxicity assay. RGD, arginine-glycyl-aspartic acid.****p<0.0001.
Fig. 3 PDL cell migration. PDL, periodontal ligament; RGD, arginine-glycyl-aspartic acid.
NS, not significant.**p<0.0001.
Fig. 4 Radiographic analysis of the alveolar bone. (A) Baseline. (B) 1 week after surgical bone defect creation. (C) Before cell-scaffold construct transplantation. (D) 4 weeks after periodontal regenerative therapy.
Fig. 5 (A) Micrograph of the regenerated periodontal tissue. B, bone; C, cementum; D, dentin;
FT, fibrous tissue; PDL, periodontal ligament. (B) Exiting alveolar bone. Note that flattened osteocytes resided in the existing alveolar
bone (arrows) adjacent to the newly regenerated bone (asterisks).
Fig. 6 CEMP-1 expression. CEMP-1, cementum protein-1; PDL, periodontal ligament; RGD, arginine-glycyl-aspartic
acid.
Fig. 7 Microcomputed tomography scan analysis of distance of alveolar bone crest (ABC) to
cement–enamel junction (CEJ). The shortest distance of alveolar bone crest and cement–enamel
junction indicated more newly formed periodontal tissue. PDL, periodontal ligament;
RGD, arginine-glycyl-aspartic acid.
Fig. 8 Micro-computed tomography scan analysis. (A) Chitosan. (B) Cell sheet-chitosan. (C) RGD-modified chitosan. (D) Cell sheet-RGD modified chitosan. RGD, arginine-glycyl-aspartic acid.
Discussion
The present study evaluated the potential of PDL cell sheet and chitosan-based materials
for periodontal tissue regeneration in the periodontal horizontal defect in M. nemestrina model. The oral conditions of M. nemestrina share many similarities with humans as well as the healing process that resembles
healing process in humans.[26] The periodontal horizontal defect was successfully created in the second incisors
by surgical approach to remove the alveolar bone surrounding the roots and in combination
with the application of elastic bands in the cervical area to induce plaque accumulation
that resembles a more natural periodontal tissue destruction process.[23] Previously, we have reported the biocompatibility and osteoconductivity properties
of chitosan in DPSCs and PDL cells.[14] The chitosan scaffold was able to stimulate the proliferation activity of these
cells. We also found that PDL cells did not express mesenchymal stromal cells (MSCs)
markers; still they have the capacity to differentiate toward osteoblastic lineage
comparable to PDL cells with MSCs markers (CD73, CD90 and CD105) (unpublished data).
Therefore, in this study we induced the heterogenous PDL cells with osteogenic supplements
for the formation of cell sheet.
Four weeks following regenerative therapy, the clinical attachment level gain was
detected in all group tested. PDL cell sheet seeded in RGD-modified chitosan showed
more clinical attachment gain; more than 50% compared with the level before regenerative
therapy was introduced. The data was consistent with the micro-CT analysis that revealed
the shortest distance between cement–enamel junction to alveolar bone crest; an evidence
for a more periodontal tissue formation was seen in the group treated with PDL cell
sheet and RGD-modified chitosan. The newly formed periodontal tissue attachment was
further analyzed by the expression of CEMP-1, the key regulator of cementogenesis.
CEMP-1 protein expression was consistently increased over the period of the periodontal
tissue regeneration process. CEMP-1 was synthesized by cementoblasts and their progenitors
in the PDL and was known to promote cementoblasts attachment, differentiation as well
as the hydroxyapatite crystals formation.[27]
[28]
The goal of periodontal regenerative therapy following the elimination of the etiology
of periodontitis is to achieve periodontal tissue regeneration.[29]
[30]
[31] The formation of new alveolar bone and cementum with the supportive PDLs restores
the periodontal tissue to its previous form and function. The amount of existing intact
bony wall will determine the regeneration process. Crater-form defect would give sufficient
mechanical and biological support to the cell-tissue construct.[32] In one-wall bone defect or horizontal bone defect cases however, periodontal tissue
regeneration process is a challenge, due to a minimal existing healthy bony wall,
lacking of vascularization and healthy cells.[4]
[5] To overcome this limitation, MSCs-based tissue engineering is believed to generate
a more predictable clinical outcome.[4]
[5]
[6]
[7]
[8]
[9] The optimal cell delivery method to the defect area is crucial to maintain cell
survival. In recent years, studies demonstrated that cell sheet provides a better
cell delivery method, as it can generate high-density cells with abundant endogenous
ECM, protects cell–cell junction, and cell surface proteins.[6]
[7]
[8]
[9] Cell sheet from various sources of MSCs has been tested for periodontal tissue regeneration.[6]
[7]
[8]
[9]
[33]
[34] Transplantation PDL cell sheet to a periodontal tissue defect model resulted in
a significant periodontal regeneration with newly formed cementum and well-oriented
PDL fibers.[33]
[34] Due to the loose structure of cell sheet, scaffold biomaterials are still essential
to support cell sheet in periodontal tissue reconstruction particularly in critical
size defect.
Biomaterial scaffold provides not only the temporary structural integrity but it also
needs to support its interaction with the cells. In the local environment, interaction
between cells and ECM has a significant impact of on cell fate for their adhesion,
proliferation, and differentiation. Mimicking this local environment would be crucial
for maintaining cells and to differentiate to a distinct phenotype of cells and to
form the desired tissues. Incorporating the adhesion ligands as biochemical elements
in biomaterials is often performed to obtain functionalized surfaces to control cell
behavior and cellular pathways. The RGD peptide sequence has been long recognized
for an essential binding motif for specific transmembrane protein that is involved
in cellular adhesion to the various ECM proteins. The addition of RGD peptide to the
chitosan scaffold for tissue regeneration was intended to support the cells adhesion
and to increase the number of infiltrated and proliferated cells and into the scaffold.[15]
[16]
[35] Various studies reported the benefit of incorporating RGD peptide on biomaterials
on osteogenic differentiation.[17]
[18]
[19]
[20] In agreement with others, our study demonstrated the potential of RGD modified-chitosan
in periodontal regeneration that reached 50% clinical attachment level gain, 4 weeks
following transplantation of PDL cell sheet and RGD-modified chitosan scaffold in
the horizontal periodontal defect. Although in the crestal areas of alveolar bone
fibrous tissues were still apparent, these cells were immunopositive for collagen
type I and osteopontin (manuscript in preparation). This indicates the osteogenic
potentials of these cells that at the later time might eventually differentiate and
form the new alveolar bone. The long-term fate of the newly regenerated tissues warrants
further studies.
Conclusion
In conclusion, horizontal periodontal defect model was successfully created in M. nemestrina model. Combination of PDL cell sheet and RGD-modified chitosan resulted in the higher
potential for periodontal tissue regeneration. The results of this study highlighted
the PDL cell sheet and RGD-modified chitosan as a promising approach for future clinical
use in periodontal regeneration.
