Bilayered construct for simultaneous regeneration of alveolar bone and periodontal ligament M. Nivedhitha Sundaram,1 S. Sowmya,1 S. Deepthi,1 Joel D. Bumgardener,2 R. Jayakumar1 1
Amrita Centre for Nanosciences and Molecular Medicine, Amrita Institute of Medical Sciences and Research Centre, Amrita Vishwa Vidyapeetham University, Kochi 682 041, India 2 Department of Biomedical Engineering, University of Memphis, Joint University of Memphis University of Tennessee, Graduate Biomedical Engineering Program, Memphis, Tennessee, USA Received 12 May 2015; accepted 15 June 2015 Published online 7 July 2015 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/jbm.b.33480 Abstract: Periodontitis is an inflammatory disease that causes destruction of tooth-supporting tissues and if left untreated leads to tooth loss. Current treatments have shown limited potential for simultaneous regeneration of the toothsupporting tissues. To recreate the complex architecture of the periodontium, we developed a bilayered construct consisting of poly(caprolactone) (PCL) multiscale electrospun membrane (to mimic and regenerate periodontal ligament, PDL) and a chitosan/2wt % CaSO4 scaffold (to mimic and regenerate alveolar bone). Scanning electron microscopy results showed the porous nature of the scaffold and formation of beadless electrospun multiscale fibers. The fiber diameter of microfiber and nanofibers was in the range of 10 6 3 mm and 377 6 3 nm, respectively. The bilayered construct showed better protein adsorption compared to the control.
Osteoblastic differentiation of human dental follicle stem cells (hDFCs) on chitosan/2wt % CaSO4 scaffold showed maximum alkaline phosphatase at seventh day followed by a decline thereafter when compared to chitosan control scaffold. Fibroblastic differentiation of hDFCs was confirmed by the expression of PLAP-1 and COL-1 proteins which were more prominent on PCL multiscale membrane in comparison to control membranes. Overall these results show that the developed bilayered construct might serve as a good candidate for the simultaneous regeneration of the alveolar bone C 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part B: and PDL. V Appl Biomater, 104B: 761–770, 2016.
Key Words: chitosan, calcium sulfate, poly(caprolactone), electrospinning, periodontal regeneration
How to cite this article: Nivedhitha Sundaram M, Sowmya S, Deepthi S, Bumgardener JD, Jayakumar R. 2016. Bilayered construct for simultaneous regeneration of alveolar bone and periodontal ligament. J Biomed Mater Res Part B 2016:104B:761–770.
INTRODUCTION
Periodontium is made up of hard tissues namely cementum and alveolar bone and soft tissues namely gingiva and periodontal ligament (PDL). Alveolar bone forms the tooth socket in which root portion of the tooth is anchored. The inorganic portion of alveolar bone is mainly composed of hydroxyapatite (HAp) and the organic portion is composed of collagen (COL) mostly type 1 and noncollagenous proteins such as osteocalcin, osteopontin, bone sialoprotein, and proteoglycans.1 PDL acts as a shock absorber that helps in anchoring the tooth to the jaw bone. It is a fibrous connective tissue composed of COL fibers type 1 and 3.2 Periodontitis is characterized by severe inflammation of the gums, loss of alveolar bone and PDL, tooth mobility, and if left untreated will lead to loss of the tooth. The major challenge in periodontal regeneration is the simultaneous restoration of the damaged hard and soft tissues, with
reconstruction of oriented PDL and its insertion into cementum on one side and alveolar bone on the other side. Current gold standard of treatment for the re-establishment of the damaged periodontium is the use of autografts that have inherent osteoinductive and osteoconductive properties. However, they are limited in quantity and might cause bleeding and infection at the donor site.3 Allografts and xenografts are also used for bone regeneration but they must undergo extensive processing conditions to minimize risk of disease transmission which reduces their osteoinductive and osteoconductive properties and hence show less clinical success.4 Guided bone regeneration (GBR) and guided tissue regeneration (GTR) are currently used for periodontal regeneration. They are resorbable or nonresorbable materials that act as barrier membranes for the prevention of epithelial migration into the defect site. The main drawbacks include poor mechanical properties and lack of effective
Correspondence to: R. Jayakumar; e-mail: [email protected] Contract grant sponsor: SERB Division, Department of Science and Technology (DST), India; contract grant number: SR/S1/OC-19/2012 Contract grant sponsor: Council of Scientific and Industrial Research (CSIR); contract grant number: 9/963(0022)2K12-EMR-I, 9/963(0034)2K13EMR-I Contract grant sponsor: Nanomission, DST, India (Thematic Unit of Excellence)
C 2015 WILEY PERIODICALS, INC. V
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regeneration capacity. Nonresorbable GBR/GTR membranes might require second surgery for removal thus increasing the risk of infection or contamination.5 Tissue engineering utilizes mediators such as biomaterials, cells, and other biochemical factors to facilitate regeneration of tissues.6 Tissue-engineered scaffolds made of chitin,7 chitosan,8 alginate,9 and many other natural polymers have been widely studied for periodontal bone regeneration. Chitosan obtained from deacetylation of chitin is a natural polymer that is recently investigated for periodontal regeneration owing to its biocompatible, biodegradable, and antimicrobial nature.10,11 It has also been reported to have mitogenic property toward osteoblastic cells by aiding their differentiation for effective bone regeneration.8 For better regeneration bioceramics such as calcium sulfate, HAp, and bioglass-based materials can be incorporated into natural or synthetic polymeric scaffolds and the composite scaffold so formed shows better osteoconductive property in comparison to the bare polymeric scaffold.12 Calcium sulfate (CaSO4), an osteoconductive material, is being used in clinics for the treatment of periodontal defect.13 It is a biodegradable material with its resorption rate comparable to the rate of new bone formation. PDL is a fibrous connective tissue wherein its regeneration necessitates an appropriate material that can easily mimic the native extracellular matrix (ECM). Electrospun membranes can satisfy this criterion and aid the regeneration process of PDL. PCL electrospun fibers with properties such as slow degradability, biocompatibility, and good mechanical strength are reported to mimic ECM of the fibrous PDL.14 A construct that can facilitate simultaneous regeneration of alveolar bone and PDL will have a significant impact for the successful regeneration of the native periodontium. Hence, our long-term goal is to develop a bilayered construct consisting of chitosan/2 wt % CaSO4 scaffold layer and PCL multiscale (micro/nano) membrane layer for the effective regeneration of lost alveolar bone and PDL tissues. This work focuses on the synthesis and characterization of a bilayered construct and the efficacy of the construct to differentiate human dental follicle stem cells (hDFCs) into osteoblastic and fibroblastic cell types that are key to the regeneration of the native periodontium. MATERIALS AND METHODS
Materials Chitosan (molecular weight: 100–150 kDa, degree of deacetylation: 85%) was purchased from Koyo Chemical, Japan. CaSO4 was purchased from Fischer Scientific, USA. PCL (molecular weight: 43,000–50,000) was purchased from Poly Sciences, Warrington, PA. Chloroform was purchased from Merck, USA. Methanol was purchased from Emplura, USA. Sodium hydroxide (NaOH) from Fisher Scientific, India. Syringe and needles used for electrospinning were purchased from BD Biosciences, India. Alamar blue reagent, trypsin-EDTA, DAPI (40 ,6-diamidino-2-phenylindole), and fetal bovine serum (FBS) were purchased from Gibco, Invitrogen Corporation, USA. Dulbecco’s-modified Eagle’s medium (DMEM), Triton X-100, paraformaldehyde (PFA),
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acetic acid, p-nitrophenylphosphate (PNPP), and dexamathasone were purchased from Sigma Aldrich, India. Alkaline phosphatase (ALP) reagent and antibiotic-antimicotic solution from Himedia, India. b-Glycerol phosphate from Fluka, Biochemica, USA. L-Ascorbic acid 2-phosphate, Sigma Aldrich, India. Fibroblast growth factor (FGF-2) was purchased from Goldbio, USA. Anti-human PLAP-1 IgG goat polyclonal antibody and FITC-labeled donkey anti-goat IgG antibody were purchased from Santa Cruz Biotechnology, India. Anti-collagen type 1 (COL-1) from Antibodies Online, USA and mouse anti-human COL-1 IgG monoclonal antibody from Thermo Scientific, USA. Preparation of chitosan/2 wt % CaSO4 scaffold 2% (w/v) Chitosan solution was prepared by dissolving chitosan powder in 1% (v/v) glacial acetic acid solution. The obtained chitosan solution was neutralized with 1N NaOH to obtain chitosan hydrogel.15 Forty milligrams of CaSO4 hemihydrate powder was added to 2 g of chitosan hydrogel. CaSO4 was thoroughly mixed with the hydrogel and lyophilized to obtain the chitosan/2 wt % CaSO4 scaffold. Preparation of PCL multiscale (micro/nano) membrane The setup used for electrospinning consists of a DC power supply (Model RR30P, 0-30 kV, Gamma High Voltage, USA), syringe pump (KD Scientific, USA), rotating mandrel (Holmarc Auto-Mechatronics, India), stationary target, and 10mL syringe with blunt 21-gauge metal needle. PCL micro (m) fibrous membrane was obtained by spinning solution of 30% PCL dissolved in chloroform, at 7 kV voltage, flow rate of 1.5 mL/h, and a tip target distance of 15 cm. PCL nano (n) fibrous membrane was obtained from a solution of 13% PCL in chloroform:methanol in the ratio 1:1, voltage of 17 kV, flow rate 1 mL/h, and a tip target distance of 20 cm. PCL multiscale (micro/nano) membrane was obtained by simultaneous spinning of PCL (m) and (n) fibers. Mandrel rotation speed of 1500 rpm was kept constant for all spinning conditions. Characterization of chitosan/2 wt % CaSO4 scaffold and PCL multiscale (micro/nano) membrane The surface morphology of the chitosan/2 wt % CaSO4 scaffold and PCL multiscale (micro/nano) membrane was examined using scanning electron microscope (JEOL JSM-6490LA Analytical SEM, Japan). Prior to imaging all samples were sputter coated with gold using a JEOL auto fine coater (model JFC-1600). The diameter of the PCL electrospun fibers was found using Image J software (n 5 3). The presence of CaSO4 in chitosan/2 wt % CaSO4 scaffold was confirmed using Fourier transform infrared (FTIR) spectrometric analysis. The spectra for chitosan, CaSO4, and PCL were confirmed using FTIR spectrometer (Shimadzu IRaffinity-1S, Japan) in the range 4000 and 400 cm21 with a resolution of 2 cm21. The presence of CaSO4 in chitosan/2 wt % CaSO4 scaffold was further confirmed using X-ray crystallography analysis (XRD) (PAN analytical X’Pert PRO X-ray diffractometer, Holland).
REGENERATION OF ALVEOLAR BONE AND PERIODONTAL LIGAMENT
ORIGINAL RESEARCH REPORT
Porosity study of the chitosan/2 wt % CaSO4 scaffold and PCL multiscale (micro/nano) membrane Liquid displacement method was used to determine the porosity of scaffold and membrane.16 The chitosan (control) and chitosan/2 wt % CaSO4 (sample) scaffolds, PCL (m), PCL (n) (control), and PCL multiscale (micro/nano) (sample) membranes of same shape and weight were used for the study (n 5 3). The diameter and width of the scaffolds and membranes were determined using a screw gauge and then the corresponding volume was calculated. The scaffolds were immersed in 100% ethanol for 48 h and the porosity of the scaffolds (p1) was calculated using the formula: p1 ¼ ½ðS22S1Þ=qV; where S1 is the weight of the scaffold before ethanol immersion, S2 the weight of scaffold after ethanol immersion, V the volume of scaffold before immersion, and q the density of ethanol. The membranes were immersed in 100% ethanol for 48 h and the porosity of the membranes (p2) was calculated using the formula: p2 ¼ ½ðE22E1Þ=qV; where E1 is the weight of membrane before ethanol immersion, E2 the weight of membrane after ethanol immersion, V the volume of membrane before immersion, and q the density of ethanol. Protein adsorption study Protein adsorption onto the scaffolds (chitosan and chitosan/2 wt % CaSO4) and membranes (PCL m, n, and micro/nano) were quantified by bicinchoninic acid (BCA) assay. Scaffolds and electrospun fibrous membranes of equal shape and weight were submerged in medium containing DMEM, 10% FBS, 100 U antibiotic-antimycotic (10% DMEM) in 96-well plate for 0.5, 2, and 6 h and incubated at 378C (n 5 3). After the incubation time period medium was removed and samples were rinsed thrice with phosphatebuffered saline (PBS) solution. BCA assay is based on the reduction of Cu21 to Cu11, which is proportional to the protein adsorbed onto the sample surface. Test scaffolds and the electrospun membranes were incubated with BCA reagent for 30 min at 378C and the absorbance was read at 562 nm. Scaffolds and the electrospun membranes incubated in serum-free medium were used as blank. Cell viability assay Human DFCs (10,000 cells) in 10% DMEM were seeded onto each scaffold and electrospun fibrous membrane and viability was evaluated at 24 and 48 h after seeding. Viability of cells was determined using the Alamar blue assay.17 Cylindrical scaffolds and membranes of 6 mm diameter and 0.4 mm height were used in the study (n 5 3/time point/ group). Optical density (OD) of the samples measured at 570 and 600 nm using a microplate reader (Biotek Power
Wave XS, USA) was recorded and compared to positive control (cells alone in 10% DMEM). Alkaline phosphatase activity ALP activity was measured to evaluate the differentiation of hDFCs to osteoblasts. Scaffolds (cylindrical scaffolds of 6 mm diameter and 0.4 mm height), chitosan and chitosan/ 2 wt % CaSO4, were placed in a 24-well plate. The hDFCs were seeded onto the scaffolds (50,000 cells/scaffold) in the presence of 10% DMEM and was incubated at 378C (n 5 3/time point/group). At 70–80% confluency, the 10% DMEM was replaced with osteogenic medium containing DMEM, 10% FBS, 100 U antibiotic-antimycotic, 10 mM bglycerophosphate, 50 mg/mL L-ascorbic acid, and 100 nM dexamethasone. The scaffolds were incubated in osteogenic medium for 7, 14, and 21 days. Osteogenic medium was replaced once in 2 days. After the determined time intervals the osteogenic medium was removed, scaffolds were rinsed with PBS, and the cell lysate was obtained by incubating cellseeded scaffolds in Triton X-100 for 2 h. The cell lysate was evaluated spectrophotometrically at 405 and 490 nm for activity of ALP enzyme via the p-nitrophenol reaction.9 Immunocytochemical staining The fibroblastic differentiation of hDFCs on PCL (m), PCL (n), and PCL multiscale (micro/nano) membrane was studied by determining the PLAP-1 and COL-1 expression by immunofluorescence. Medium containing 10% DMEM along with 20 ng/mL FGF-2 was used as the fibroblast differentiation medium.18 The hDFCs were seeded (40,000 cells/membrane) onto the electrospun fibrous membrane (cylindrical scaffolds of 6 mm diameter and 0.4 mm height) and incubated in fibroblast differentiation medium (n 5 3/time point/group). At 7 and 14 days cells were evaluated for PLAP-1 expression and 14 and 21 days for COL-1 expression. For staining, cells were fixed using 4% PFA, permeabilized by 0.5% Triton-X-100, and blocked using 1% FBS. Next, primary antibodies, goat (polyclonal) anti-human PLAP-1 IgG, and mouse monoclonal anti-human COL-1 IgG were added to cells on the membranes as per the manufacture’s recommendations and kept overnight at 48C. Following PBS wash, the membranes were incubated with secondary antibodies, FITC-labeled donkey anti-goat IgG, and FITC-labeled goat anti-mouse IgG for 1 h at room temperature as per manufacturer’s instructions. All membranes were then stained with DAPI and analyzed under confocal microscopy (Leica TCS SPS II, Germany). Image J software was used to quantify cumulative total cell fluorescence (CTCF) from the fluorescence images of the antibody-labeled cells on membranes by using the formula: CTCF ¼ Integrated density2ðArea of selected cell 3 Mean fluorescence of background readingsÞ:
Statistical analysis Results of all samples (n 5 3 per group) examined were computed as mean 6 standard deviation. Statistical analysis
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FIGURE 1. Pictorial representation of bilayered construct: PCL multiscale (micro/nano) membrane (top layer) and chitosan/2 wt % CaSO4 scaffold (bottom layer).
FIGURE 2. SEM Images of (A) chitosan scaffold, (B) chitosan/2 wt % CaSO4 scaffold, inset shows the presence of needle-shaped CaSO4 in the scaffold, (C) XRD spectrum of (a) chitosan, (b) chitosan/2 wt % CaSO4 scaffolds, and (c) CaSO4 powder, (D) FTIR spectrum of (a) CaSO4 powder, (b) chitosan, and (c) chitosan/2 wt % CaSO4 scaffolds.
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REGENERATION OF ALVEOLAR BONE AND PERIODONTAL LIGAMENT
ORIGINAL RESEARCH REPORT
FIGURE 3. SEM images of membranes (A) PCL (m), (B) PCL (n), and (C) PCL multiscale (micro/nano) fibers. (D) FTIR spectrum of (a) PCL (m), (b) PCL (n), and (c) PCL multiscale (micro/nano) membranes.
was performed using Student’s two-tailed t-test and ANOVA. p-Values of
Amrita Centre for Nanosciences and Molecular Medicine, Amrita Institute of Medical Sciences and Research Centre, Amrita Vishwa Vidyapeetham University, Kochi 682 041, India 2 Department of Biomedical Engineering, University of Memphis, Joint University of Memphis University of Tennessee, Graduate Biomedical Engineering Program, Memphis, Tennessee, USA Received 12 May 2015; accepted 15 June 2015 Published online 7 July 2015 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/jbm.b.33480 Abstract: Periodontitis is an inflammatory disease that causes destruction of tooth-supporting tissues and if left untreated leads to tooth loss. Current treatments have shown limited potential for simultaneous regeneration of the toothsupporting tissues. To recreate the complex architecture of the periodontium, we developed a bilayered construct consisting of poly(caprolactone) (PCL) multiscale electrospun membrane (to mimic and regenerate periodontal ligament, PDL) and a chitosan/2wt % CaSO4 scaffold (to mimic and regenerate alveolar bone). Scanning electron microscopy results showed the porous nature of the scaffold and formation of beadless electrospun multiscale fibers. The fiber diameter of microfiber and nanofibers was in the range of 10 6 3 mm and 377 6 3 nm, respectively. The bilayered construct showed better protein adsorption compared to the control.
Osteoblastic differentiation of human dental follicle stem cells (hDFCs) on chitosan/2wt % CaSO4 scaffold showed maximum alkaline phosphatase at seventh day followed by a decline thereafter when compared to chitosan control scaffold. Fibroblastic differentiation of hDFCs was confirmed by the expression of PLAP-1 and COL-1 proteins which were more prominent on PCL multiscale membrane in comparison to control membranes. Overall these results show that the developed bilayered construct might serve as a good candidate for the simultaneous regeneration of the alveolar bone C 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part B: and PDL. V Appl Biomater, 104B: 761–770, 2016.
Key Words: chitosan, calcium sulfate, poly(caprolactone), electrospinning, periodontal regeneration
How to cite this article: Nivedhitha Sundaram M, Sowmya S, Deepthi S, Bumgardener JD, Jayakumar R. 2016. Bilayered construct for simultaneous regeneration of alveolar bone and periodontal ligament. J Biomed Mater Res Part B 2016:104B:761–770.
INTRODUCTION
Periodontium is made up of hard tissues namely cementum and alveolar bone and soft tissues namely gingiva and periodontal ligament (PDL). Alveolar bone forms the tooth socket in which root portion of the tooth is anchored. The inorganic portion of alveolar bone is mainly composed of hydroxyapatite (HAp) and the organic portion is composed of collagen (COL) mostly type 1 and noncollagenous proteins such as osteocalcin, osteopontin, bone sialoprotein, and proteoglycans.1 PDL acts as a shock absorber that helps in anchoring the tooth to the jaw bone. It is a fibrous connective tissue composed of COL fibers type 1 and 3.2 Periodontitis is characterized by severe inflammation of the gums, loss of alveolar bone and PDL, tooth mobility, and if left untreated will lead to loss of the tooth. The major challenge in periodontal regeneration is the simultaneous restoration of the damaged hard and soft tissues, with
reconstruction of oriented PDL and its insertion into cementum on one side and alveolar bone on the other side. Current gold standard of treatment for the re-establishment of the damaged periodontium is the use of autografts that have inherent osteoinductive and osteoconductive properties. However, they are limited in quantity and might cause bleeding and infection at the donor site.3 Allografts and xenografts are also used for bone regeneration but they must undergo extensive processing conditions to minimize risk of disease transmission which reduces their osteoinductive and osteoconductive properties and hence show less clinical success.4 Guided bone regeneration (GBR) and guided tissue regeneration (GTR) are currently used for periodontal regeneration. They are resorbable or nonresorbable materials that act as barrier membranes for the prevention of epithelial migration into the defect site. The main drawbacks include poor mechanical properties and lack of effective
Correspondence to: R. Jayakumar; e-mail: [email protected] Contract grant sponsor: SERB Division, Department of Science and Technology (DST), India; contract grant number: SR/S1/OC-19/2012 Contract grant sponsor: Council of Scientific and Industrial Research (CSIR); contract grant number: 9/963(0022)2K12-EMR-I, 9/963(0034)2K13EMR-I Contract grant sponsor: Nanomission, DST, India (Thematic Unit of Excellence)
C 2015 WILEY PERIODICALS, INC. V
761
regeneration capacity. Nonresorbable GBR/GTR membranes might require second surgery for removal thus increasing the risk of infection or contamination.5 Tissue engineering utilizes mediators such as biomaterials, cells, and other biochemical factors to facilitate regeneration of tissues.6 Tissue-engineered scaffolds made of chitin,7 chitosan,8 alginate,9 and many other natural polymers have been widely studied for periodontal bone regeneration. Chitosan obtained from deacetylation of chitin is a natural polymer that is recently investigated for periodontal regeneration owing to its biocompatible, biodegradable, and antimicrobial nature.10,11 It has also been reported to have mitogenic property toward osteoblastic cells by aiding their differentiation for effective bone regeneration.8 For better regeneration bioceramics such as calcium sulfate, HAp, and bioglass-based materials can be incorporated into natural or synthetic polymeric scaffolds and the composite scaffold so formed shows better osteoconductive property in comparison to the bare polymeric scaffold.12 Calcium sulfate (CaSO4), an osteoconductive material, is being used in clinics for the treatment of periodontal defect.13 It is a biodegradable material with its resorption rate comparable to the rate of new bone formation. PDL is a fibrous connective tissue wherein its regeneration necessitates an appropriate material that can easily mimic the native extracellular matrix (ECM). Electrospun membranes can satisfy this criterion and aid the regeneration process of PDL. PCL electrospun fibers with properties such as slow degradability, biocompatibility, and good mechanical strength are reported to mimic ECM of the fibrous PDL.14 A construct that can facilitate simultaneous regeneration of alveolar bone and PDL will have a significant impact for the successful regeneration of the native periodontium. Hence, our long-term goal is to develop a bilayered construct consisting of chitosan/2 wt % CaSO4 scaffold layer and PCL multiscale (micro/nano) membrane layer for the effective regeneration of lost alveolar bone and PDL tissues. This work focuses on the synthesis and characterization of a bilayered construct and the efficacy of the construct to differentiate human dental follicle stem cells (hDFCs) into osteoblastic and fibroblastic cell types that are key to the regeneration of the native periodontium. MATERIALS AND METHODS
Materials Chitosan (molecular weight: 100–150 kDa, degree of deacetylation: 85%) was purchased from Koyo Chemical, Japan. CaSO4 was purchased from Fischer Scientific, USA. PCL (molecular weight: 43,000–50,000) was purchased from Poly Sciences, Warrington, PA. Chloroform was purchased from Merck, USA. Methanol was purchased from Emplura, USA. Sodium hydroxide (NaOH) from Fisher Scientific, India. Syringe and needles used for electrospinning were purchased from BD Biosciences, India. Alamar blue reagent, trypsin-EDTA, DAPI (40 ,6-diamidino-2-phenylindole), and fetal bovine serum (FBS) were purchased from Gibco, Invitrogen Corporation, USA. Dulbecco’s-modified Eagle’s medium (DMEM), Triton X-100, paraformaldehyde (PFA),
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acetic acid, p-nitrophenylphosphate (PNPP), and dexamathasone were purchased from Sigma Aldrich, India. Alkaline phosphatase (ALP) reagent and antibiotic-antimicotic solution from Himedia, India. b-Glycerol phosphate from Fluka, Biochemica, USA. L-Ascorbic acid 2-phosphate, Sigma Aldrich, India. Fibroblast growth factor (FGF-2) was purchased from Goldbio, USA. Anti-human PLAP-1 IgG goat polyclonal antibody and FITC-labeled donkey anti-goat IgG antibody were purchased from Santa Cruz Biotechnology, India. Anti-collagen type 1 (COL-1) from Antibodies Online, USA and mouse anti-human COL-1 IgG monoclonal antibody from Thermo Scientific, USA. Preparation of chitosan/2 wt % CaSO4 scaffold 2% (w/v) Chitosan solution was prepared by dissolving chitosan powder in 1% (v/v) glacial acetic acid solution. The obtained chitosan solution was neutralized with 1N NaOH to obtain chitosan hydrogel.15 Forty milligrams of CaSO4 hemihydrate powder was added to 2 g of chitosan hydrogel. CaSO4 was thoroughly mixed with the hydrogel and lyophilized to obtain the chitosan/2 wt % CaSO4 scaffold. Preparation of PCL multiscale (micro/nano) membrane The setup used for electrospinning consists of a DC power supply (Model RR30P, 0-30 kV, Gamma High Voltage, USA), syringe pump (KD Scientific, USA), rotating mandrel (Holmarc Auto-Mechatronics, India), stationary target, and 10mL syringe with blunt 21-gauge metal needle. PCL micro (m) fibrous membrane was obtained by spinning solution of 30% PCL dissolved in chloroform, at 7 kV voltage, flow rate of 1.5 mL/h, and a tip target distance of 15 cm. PCL nano (n) fibrous membrane was obtained from a solution of 13% PCL in chloroform:methanol in the ratio 1:1, voltage of 17 kV, flow rate 1 mL/h, and a tip target distance of 20 cm. PCL multiscale (micro/nano) membrane was obtained by simultaneous spinning of PCL (m) and (n) fibers. Mandrel rotation speed of 1500 rpm was kept constant for all spinning conditions. Characterization of chitosan/2 wt % CaSO4 scaffold and PCL multiscale (micro/nano) membrane The surface morphology of the chitosan/2 wt % CaSO4 scaffold and PCL multiscale (micro/nano) membrane was examined using scanning electron microscope (JEOL JSM-6490LA Analytical SEM, Japan). Prior to imaging all samples were sputter coated with gold using a JEOL auto fine coater (model JFC-1600). The diameter of the PCL electrospun fibers was found using Image J software (n 5 3). The presence of CaSO4 in chitosan/2 wt % CaSO4 scaffold was confirmed using Fourier transform infrared (FTIR) spectrometric analysis. The spectra for chitosan, CaSO4, and PCL were confirmed using FTIR spectrometer (Shimadzu IRaffinity-1S, Japan) in the range 4000 and 400 cm21 with a resolution of 2 cm21. The presence of CaSO4 in chitosan/2 wt % CaSO4 scaffold was further confirmed using X-ray crystallography analysis (XRD) (PAN analytical X’Pert PRO X-ray diffractometer, Holland).
REGENERATION OF ALVEOLAR BONE AND PERIODONTAL LIGAMENT
ORIGINAL RESEARCH REPORT
Porosity study of the chitosan/2 wt % CaSO4 scaffold and PCL multiscale (micro/nano) membrane Liquid displacement method was used to determine the porosity of scaffold and membrane.16 The chitosan (control) and chitosan/2 wt % CaSO4 (sample) scaffolds, PCL (m), PCL (n) (control), and PCL multiscale (micro/nano) (sample) membranes of same shape and weight were used for the study (n 5 3). The diameter and width of the scaffolds and membranes were determined using a screw gauge and then the corresponding volume was calculated. The scaffolds were immersed in 100% ethanol for 48 h and the porosity of the scaffolds (p1) was calculated using the formula: p1 ¼ ½ðS22S1Þ=qV; where S1 is the weight of the scaffold before ethanol immersion, S2 the weight of scaffold after ethanol immersion, V the volume of scaffold before immersion, and q the density of ethanol. The membranes were immersed in 100% ethanol for 48 h and the porosity of the membranes (p2) was calculated using the formula: p2 ¼ ½ðE22E1Þ=qV; where E1 is the weight of membrane before ethanol immersion, E2 the weight of membrane after ethanol immersion, V the volume of membrane before immersion, and q the density of ethanol. Protein adsorption study Protein adsorption onto the scaffolds (chitosan and chitosan/2 wt % CaSO4) and membranes (PCL m, n, and micro/nano) were quantified by bicinchoninic acid (BCA) assay. Scaffolds and electrospun fibrous membranes of equal shape and weight were submerged in medium containing DMEM, 10% FBS, 100 U antibiotic-antimycotic (10% DMEM) in 96-well plate for 0.5, 2, and 6 h and incubated at 378C (n 5 3). After the incubation time period medium was removed and samples were rinsed thrice with phosphatebuffered saline (PBS) solution. BCA assay is based on the reduction of Cu21 to Cu11, which is proportional to the protein adsorbed onto the sample surface. Test scaffolds and the electrospun membranes were incubated with BCA reagent for 30 min at 378C and the absorbance was read at 562 nm. Scaffolds and the electrospun membranes incubated in serum-free medium were used as blank. Cell viability assay Human DFCs (10,000 cells) in 10% DMEM were seeded onto each scaffold and electrospun fibrous membrane and viability was evaluated at 24 and 48 h after seeding. Viability of cells was determined using the Alamar blue assay.17 Cylindrical scaffolds and membranes of 6 mm diameter and 0.4 mm height were used in the study (n 5 3/time point/ group). Optical density (OD) of the samples measured at 570 and 600 nm using a microplate reader (Biotek Power
Wave XS, USA) was recorded and compared to positive control (cells alone in 10% DMEM). Alkaline phosphatase activity ALP activity was measured to evaluate the differentiation of hDFCs to osteoblasts. Scaffolds (cylindrical scaffolds of 6 mm diameter and 0.4 mm height), chitosan and chitosan/ 2 wt % CaSO4, were placed in a 24-well plate. The hDFCs were seeded onto the scaffolds (50,000 cells/scaffold) in the presence of 10% DMEM and was incubated at 378C (n 5 3/time point/group). At 70–80% confluency, the 10% DMEM was replaced with osteogenic medium containing DMEM, 10% FBS, 100 U antibiotic-antimycotic, 10 mM bglycerophosphate, 50 mg/mL L-ascorbic acid, and 100 nM dexamethasone. The scaffolds were incubated in osteogenic medium for 7, 14, and 21 days. Osteogenic medium was replaced once in 2 days. After the determined time intervals the osteogenic medium was removed, scaffolds were rinsed with PBS, and the cell lysate was obtained by incubating cellseeded scaffolds in Triton X-100 for 2 h. The cell lysate was evaluated spectrophotometrically at 405 and 490 nm for activity of ALP enzyme via the p-nitrophenol reaction.9 Immunocytochemical staining The fibroblastic differentiation of hDFCs on PCL (m), PCL (n), and PCL multiscale (micro/nano) membrane was studied by determining the PLAP-1 and COL-1 expression by immunofluorescence. Medium containing 10% DMEM along with 20 ng/mL FGF-2 was used as the fibroblast differentiation medium.18 The hDFCs were seeded (40,000 cells/membrane) onto the electrospun fibrous membrane (cylindrical scaffolds of 6 mm diameter and 0.4 mm height) and incubated in fibroblast differentiation medium (n 5 3/time point/group). At 7 and 14 days cells were evaluated for PLAP-1 expression and 14 and 21 days for COL-1 expression. For staining, cells were fixed using 4% PFA, permeabilized by 0.5% Triton-X-100, and blocked using 1% FBS. Next, primary antibodies, goat (polyclonal) anti-human PLAP-1 IgG, and mouse monoclonal anti-human COL-1 IgG were added to cells on the membranes as per the manufacture’s recommendations and kept overnight at 48C. Following PBS wash, the membranes were incubated with secondary antibodies, FITC-labeled donkey anti-goat IgG, and FITC-labeled goat anti-mouse IgG for 1 h at room temperature as per manufacturer’s instructions. All membranes were then stained with DAPI and analyzed under confocal microscopy (Leica TCS SPS II, Germany). Image J software was used to quantify cumulative total cell fluorescence (CTCF) from the fluorescence images of the antibody-labeled cells on membranes by using the formula: CTCF ¼ Integrated density2ðArea of selected cell 3 Mean fluorescence of background readingsÞ:
Statistical analysis Results of all samples (n 5 3 per group) examined were computed as mean 6 standard deviation. Statistical analysis
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FIGURE 1. Pictorial representation of bilayered construct: PCL multiscale (micro/nano) membrane (top layer) and chitosan/2 wt % CaSO4 scaffold (bottom layer).
FIGURE 2. SEM Images of (A) chitosan scaffold, (B) chitosan/2 wt % CaSO4 scaffold, inset shows the presence of needle-shaped CaSO4 in the scaffold, (C) XRD spectrum of (a) chitosan, (b) chitosan/2 wt % CaSO4 scaffolds, and (c) CaSO4 powder, (D) FTIR spectrum of (a) CaSO4 powder, (b) chitosan, and (c) chitosan/2 wt % CaSO4 scaffolds.
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REGENERATION OF ALVEOLAR BONE AND PERIODONTAL LIGAMENT
ORIGINAL RESEARCH REPORT
FIGURE 3. SEM images of membranes (A) PCL (m), (B) PCL (n), and (C) PCL multiscale (micro/nano) fibers. (D) FTIR spectrum of (a) PCL (m), (b) PCL (n), and (c) PCL multiscale (micro/nano) membranes.
was performed using Student’s two-tailed t-test and ANOVA. p-Values of
