Journal of Biomaterials Applications

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Engineering bone tissue using human dental pulp stem cells and an osteogenic collagen-hydroxyapatite-poly(?-lactide-co- ε-caprolactone) scaffold Adil Akkouch, Ze Zhang and Mahmoud Rouabhia J Biomater Appl published online 2 May 2013 DOI: 10.1177/0885328213486705

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Original Article

Engineering bone tissue using human dental pulp stem cells and an osteogenic collagen-hydroxyapatite-poly (L-lactide-co-"-caprolactone) scaffold

Journal of Biomaterials Applications 0(0) 1–15 ! The Author(s) 2013 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav DOI: 10.1177/0885328213486705 jba.sagepub.com

Adil Akkouch1,2, Ze Zhang2 and Mahmoud Rouabhia1

Abstract The aim of this study was to design a new natural/synthetic bioactive bone scaffold for potential use in bone replacement applications. We developed a tri-component osteogenic composite scaffold made of collagen (Coll), hydroxyapatite (HA) and poly(L-lactide-co-"-caprolactone) (PLCL). This Coll/HA/PLCL composite scaffold was combined with human osteoblast-like cells obtained by differentiation of dental pulp stem cells (DPSCs) to engineer bone tissue in vitro. Results show that the 3D Coll/HA/PLCL composite scaffold was highly porous, thereby enabling osteoblast-like cell adhesion and growth. Cultured in the Coll/HA/PLCL scaffold, the osteoblast-like cells expressed different osteogenic genes, produced alkaline phosphatase and formed nodules more than did PLCL alone. Micro-CT analyses revealed a significant (30%) increase of tissue mineralisation on the surface as well as inside of the Coll/HA/PLCL scaffold, thus confirming its effectiveness as a bone regeneration platform. Keywords Human dental pulp stem cells, differentiation, biomaterial, poly(lactide-co-"-caprolactone), collagen, hydroxyapatite, bone regeneration

Introduction Millions of patients worldwide suffer from bone defects resulting from trauma, congenital abnormalities, cancer resection or progressive deforming diseases.1 It is estimated that 5–10% of these patients fail to heal properly, due to non-union or delayed union bone repair.2 In the case of periodontal disease, nearly half of the adults between the ages of 45 and 65 suffer from periodontal disease resulting in alveolar bone defects that lead to a loss of almost 6.5 teeth per individual and often insufficient bone mass to support dental implants.3 Clearly, deformities caused by bone defects reduce key functions and generally affect patient health. Repairing bone defects using bone grafts may represent complex challenges. For example, the autologous bone graft is limited by its size and availability and requires a second surgical operation, while the allograft may display immunogenicity.4 The search therefore continues for safe and effective ways to repair bone defects. In recent years, bone tissue engineering has emerged as a highly promising approach to develop biologically

active bone substitutes to restore, maintain and improve bone tissue function.1–3 In light of the aging population and the worldwide lack of donor tissue, various stem cells have been considered as a potentially powerful tool for bone tissue engineering combined with an appropriate scaffold to generate bone tissue that mimics natural bone.1,2 A bone-regenerating scaffold should ideally be osteoconductive, possess a 3D porous structure and be biodegradable to match the formation of a new bone.5–7 In the last decade, several novel processing techniques have been developed using synthetic polymers to produce scaffolds. Among these is

1

Groupe de recherche en ećologie buccale, Faculty of Dentistry, Laval University, Quebec, Canada 2 Department of Surgery, Laval University and Saint-François d’Assise Hospital Research Centre, CHUQ, Quebec, Canada Corresponding author: Mahmoud Rouabhia, Groupe de Recherche en Ećologie Buccale, Faculte´ de Me´decine Dentaire, Universite´ Laval, Que´bec, QC, Canada. Email: [email protected]



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poly(L-lactide-co-"-caprolactone) (PLCL), a copolymer made of randomly polymerised lactide and "-caprolactone polymers. PLCL has a good track record in such medical applications as vascular grafts and for periodontal regeneration.8,9 The most attractive property of PLCL, compared to other synthetic biodegradable polymers such as polylactide and PLCL, is its elasticity, which is inherent to most natural bone.8,9 However, PLCL has a relatively low affinity for osteoblasts.10 In a biological system, cells are in contact with the extracellular matrix (ECM) and the surrounding environment that contribute to cell adhesion, proliferation and bone remodelling by the transduction of biological, biochemical and physical signals.1,2 Because collagen type I and hydroxyapatite (HA) are the key components of bone ECM,11 an in vitro-designed bone scaffold must ideally contain these two components in order to mimic the natural environment of bone. Engineering tissues require cells that proliferate and differentiate into the required phenotype.12 Mesenchymal stem cells (MSCs) are capable of regenerating damaged tissue13 and differentiating into osteoblasts, chondrocytes and adipocytes.12,14 MSCs are primarily isolated from bone marrow, umbilical cord blood, adipose tissue and muscles, as well as from the heart and the dermis.15,16 Another accessible site to isolate stem cells is in dental pulp. Dental pulp stem cells (DPSCs) in human adult permanent teeth12,17 are easily accessible, are available in large quantities with a high proliferative rate and can be cryopreserved for long periods.18,19 DPSCs can undergo osteogenic, dentinogenic, chondrogenic, adipogenic and even neuronal and myogenic differentiation.20–23 However, few studies have investigated the regenerative potential of osteoblasts derived from human DPSCs combined with a biodegradable, porous and malleable bone scaffold for guided bone regeneration. The goal of this study was thus to differentiate human DPSCs into osteoblast-like cells and to investigate the interaction of these osteoblast-like cells with a new malleable composite scaffold made of collagen (Coll), HA, and PLCL components for bone tissue regeneration. Previously published work24,25 focused on electrospinning technology because it generated scaffolds made of nanofibres and could incorporate bioactive components such as Coll, gelatine, hyaluronic acid and HA during the electrospinning process. Despite the advantages of electrospun scaffolds, they have nano-sized pores and low compression strength, thus ultimately limiting cell infiltration within the scaffold. Furthermore, the mechanical properties of electrospun nanofibre scaffolds do not match those of bone.24,25 Our approach is therefore unique, not because of the scaffold’s composition but rather its balanced design: the bioactive components, the

relatively strong and malleable mechanical properties, the featured porous structure with macrochannels and the use of human DPSC-derived osteoblasts. This novel scaffold may therefore promote osteogenic cell adhesion and growth leading to the formation of bone tissue for clinical applications.

Experimental procedures Scaffold fabrication Using a biomimetic strategy combined with freezedrying and salt leaching techniques, we developed a tri-component 3D scaffold consisting of biodegradable elastic PLCL (70 : 30) copolymer (PURAC Biochem, Gorinchem, The Netherlands), natural type I Coll (Sigma–Aldrich, Oakville, ON, Canada) biopolymer, and bioactive mineral HA (Sigma–Aldrich). Briefly, the Coll/HA/PLCL scaffold was prepared following a two-step process. We first produced mineralised type I collagen with hydroxyapatite (Coll/HA) by means of a self-assembling method. To do so, HA powder was added to a collagenous solution (10 mg/ml) at a ratio of 30 : 70 under stirring (350 r/min) at room temperature for 4 h. The Coll/HA mixture was then titrated with a 1 M sodium hydroxide solution (MAT, Beauport, QC, Canada) to adjust the pH to 7.4, after which time the mixture was centrifuged at 1200 r/min for 10 min and subsequently dried for 24 h at room temperature. In step 2, the dried Coll/HA mixture was added to a 5% (wt/v) PLCL (70 : 30) solution in 1,4-dioxane (Fisher Scientific, Ottawa, ON, Canada) and was stirred at 450 r/min at room temperature. To produce a porous scaffold, sieved NaCl particles of 75 to 125 mm in diameter (MAT) were used at a 9 : 1 ratio by weight. Following homogenisation, the Coll/HA/ PLCL solution was poured into a cylindrical mould (height 5.5 cm and radius 0.75 cm) and frozen thereafter in ice water. The moulds were then lyophilised overnight to remove any remaining solvent. Porogen leaching was performed in distilled water at room temperature. The water was changed three times per day for 3 days, after which time the Coll/HA/PLCL porous 3D scaffolds were dried in air and stored in a desiccator until use. The porous PLCL scaffolds were prepared similarly to the controls.

Human dental pulp stem cell isolation, expansion and phenotyping Human third molars were obtained from adult patients (18–25 years old, six donors) in accordance with Laval University Ethics Committee guidelines. The teeth were extracted under local anaesthesia with the consent of the patient. Following extraction, the teeth were kept



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on ice in phosphate-buffered saline (PBS) and were cleaned with a PBS solution containing antibiotics. A 0.5–1.0 mm-deep groove was cut around the circumference of the teeth using a sterile hand-held high-speed drill at the cemento-enamel junction level. The pulp tissue of each tooth was then extracted with endodontic files. The extracted pulp tissue was subsequently digested in collagenase type I (3 mg/ml, Invitrogen Life Technologies, Burlington, ON, Canada) solution for 1 h at 37 C. The extracted cells were cultured in a-MEM culture medium (Invitrogen Life Technologies) supplemented with 10% inactivated fetal calf serum (FBS, NCS, fetal clone II; Hyclone, Logan, UT), antibiotics (200 U/ml of penicillin, 200 mg/ml of streptomicin) (Schering, Pointe-Claire, QC, Canada) and 2.5 mg/ml of amphotericin B (Sigma-Aldrich). The grown cells were then trypsinised and re-plated for expansion. Cell phenotyping was performed at passages 4–5 using specific antibodies CD73, CD90, CD105, CD34, CD45, CD11b and CD19, and class II human leukocyte antigen (HLA-DR) (BD Biosciences, San Jose, CA).

Human dental pulp stem cell culture and osteogenic differentiation To differentiate human DPSCs into osteoblasts, passages 4–6 of the expanded human DPSCs were seeded at 5 105 cells/well in a 5.5 cm Petri dish and were cultured in normal or osteoblast differentiating culture medium. This differentiation medium consisted of a-MEM medium supplemented with 20% FBS, 10 mM of b-glycerophosphate, 0.5 mM of ascorbic acid and 107 M of dexamethasone.26 For the controls, DPSCs were cultured in normal non-osteoblast differentiating medium which consisted of a-MEM medium supplemented with 20% FBS. The cultures were maintained for three weeks, with medium changed every 24 h, and were subjected thereafter to Alizarin Red staining (ARS), cetylpyridinium chloride (CPC) analyses, then mineralization was normalised to the number of viable cells. Runx2 gene expression studies were also performed to confirm the differentiation of DPSC to osteoblast-like cells.

ARS and semi-quantitative CPC extraction Following culture for three weeks under differentiation and non-differentiation conditions, the DPSCs were fixed with 4% paraformaldehyde for 15 min. After washing with deionised water, the cells were stained with ARS (40 mM) for 20 min at room temperature. Following three washes with dH2O, the stained specimens were observed under an inverted microscope to detect the presence of mineralised matrix. The stain was then extracted using 10% CPC buffer for 1 h with

gentle shaking, and the absorbance at 550 nm was read by means of a spectrophotometric plate reader (xMark, BioRad Laboratories, Mississauga, ON, Canada).

Alkaline phosphatase activity After three weeks of cell differentiation, the alkaline phosphatase activity was assessed by using p-nitrophenyl phosphate disodium salt (PNPP, Phosphatase Substrate Kit, No. 37620, Pierce Biotechnology, Rockford, IL) as the substrate. Briefly, 400 l of PNPP solution was incubated with 400 l of cell lysate in a 96-well plate at room temperature for 30 min. This was followed by adding 200 l of 2 N NaOH to stop the conversion of p-nitrophenol into p-nitrophenylate. A BioRad microplate reader (xMark) was used to measure the amount of light absorbance at a wavelength of 405 nm. Comparable samples were digested with trypsin and released cells were counted using trypan blue assay to determine viable cells. Results were normalised to a determined number of viable cells.

Evaluation of RUNX-2 gene expression by quantitative real time RT-PCR Total RNA was isolated from osteoblast-like cells by means of the Illustra RNAspin Mini kit (GE Healthcare UK Ltd., Buckingham, UK). Reverse transcription was performed by using 1 mg of total RNA in a first-strand cDNA synthesis reaction with Moloney murine leukemia virus (M-MLV) reverse transcriptase (Invitrogen) and random hexamers (Applied Biosystems Inc., Foster City, CA). The amount of mRNA transcripts was measured by means of a BioRad CFX96 real-time PCR detection system (BioRad). Reactions were performed with PCR Supermix from Bio-Rad (iQ SYBR Green Supermix). Primers were added to the reaction mix at a final concentration of 200 nM (Table 1). Five ml of each cDNA sample were added to a 20 ml PCR mixture containing 12.5 ml of iQ SYBR Green Supermix (Bio-Rad), 0.5 ml of specific primers for RUNX-2 and GAPDH (Medicorp Inc., Montreál, QC, Canada) and 7 ml of RNase/DNasefree water (MP Biomedicals, Solon, OH). The reactions were carried out in a MyCyclerTM Thermal Cycler (BioRad). For the qPCR, the CT was automatically determined using the accompanying Bio-Rad CFX manager. RUNX-2 mRNA was amplified by PCR with a maximum of 30 cycles, and GAPDH mRNA was amplified by PCR with a maximum of 16 cycles. The specificity of each primer pair was assessed by the presence of a single melting temperature peak. The GAPDH produced uniform expression levels, varying by less than



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Journal of Biomaterials Applications 0(0) Table 1. Primers used for quantitative polymerase chain reaction (qRT-PCR) analyses. Gene Name

GenBank No.

Primers sequences

RUNX2

NM_001024630

Oteocalcin

NM_199173

Osterix

AF477981

RUNKL

AF019047

GAPDH

NM_002046

Forward: 50 -AAC CCA CGA ATG CAC TAT CCA-30 Reverse: 50 -CGG ACA TAC CGA GGG ACA TG-30 Forward: 50 -TAGTGAAGAGACCCAGGCGC-30 Reverse: 50 -CACAGTCCGGATTGAGCTCA-30 Forward: 50 -CCCCACCTCTTGCAACCA-30 Reverse: 50 -CCTTCT AGCTGCCCACTATTTCC-30 Forward: 50 -TGATTCATGTAGGAGAATTAAACAGG-30 Reverse: 50 -GATGTGCTGTGATCCAACGA-30 Forward: 50 -GGTATCGTCGAAGGACTCATGAC-30 Reverse: 50 -ATGCCAGTGAGCTTCCCGTTCAGC-30

0.5 CTs between sample conditions, and was therefore used as a reference gene for this study.

Culture of human osteoblast-like cells into Coll/HA/ PLCL scaffolds Cylindrical Coll/HA/PLCL and PLCL (control) scaffolds were cut into disks (diameter 10 mm and thickness 2 mm), sterilised with 70% ethanol for 30 min, and subsequently washed twice with sterile deionised water. The specimens were then placed in a 24-well tissue culture plate and incubated in a-MEM culture medium supplemented with 1% (v/v) penicillin/streptomycin at 37 C overnight (preconditioning). Cell seeding was performed by adding osteoblast-like cell suspension dropwise to ensure cell loading directly into the scaffolds. Specifically, 50 l of medium containing 5 105 cells were directly layered onto each scaffold surface and incubated in a humidified incubator for 1 h. The wells were then flooded with 1 ml of medium and cultured under standard static conditions at 37 C and 5% CO2 for various culture periods (8 and 24 h, 4, 7, 14 and 21 days). Following each culture period, the ALP activity and tissue mineralisation were analysed as described above. These were supported by osteogenic gene’s expression to confirm the osteoblast phenotype following culture in the Coll/HA/PLCL scaffold.

Gene’s expression by the osteoblast-like cells following culture in Coll/HA/PLCL scaffold RNA extraction and quantification Osteoblast-like cells were cultured into the Coll/HA/ PLCL or PLCL scaffold for seven days, then used to evaluate the expression of oteogenic genes that include Runx2, Osterix, osteocalcin and RUNKL. Total cellular RNA was extracted as described above and was used to perform qRT-PCR. The RNA (1 mg of each

specimen) was reverse transcripted into cDNA using M-MLV reverse transcriptase (Invitrogen) and random hexamers (Applied Biosystems, Foster City, CA). The RT conditions were 10 min at 65 C, 1 h at 37 C and 10 min at 65 C. The amount of mRNA transcripts was measured by means of a Bio-Rad CFX96 real-time PCR detection system (Bio-Rad, Mississauga, ON, Canada). Reactions were performed using a PCR Supermix from Bio-Rad (iQ SYBR Green Supermix). Primers were added to the reaction mix at a final concentration of 200 nM (Table 1). Five microlitres of each cDNA sample were added to a 20 -ml PCR mixture containing 12.5 ml of iQ SYBR Green Supermix (Bio-Rad), 0.5 ml of specific primers for osteocalcin and ALP (Medicorp, Montreál, QC, Canada), and 7 ml of Rnase/Dnase-free water (MP Biomedicals, Solon, OH). Reaction was performed in a MyCyclerTM Thermal Cycler (Bio-Rad) as described above.

Scanning electron microscopy Scanning electron microscopy (SEM) was used to monitor cell attachment, morphology and mineral production by the osteoblast-like cells. At the scheduled time points, the cells were fixed in ethylene glycol for 15 min and then dehydrated in a vacuum oven at room temperature. Samples were then sputter-coated with gold and viewed under a JEOL 6360 LV SEM (Soquelec Inc., Montreál, QC, Canada) at an accelerating voltage of 15 kV.

Hoechst staining Following each culture period, specimens were washed and fixed with 25% glacial acetic acid in a methanol solution (v/v). Each well was then supplemented with 0.5 ml of Hoechst 33342 (H42) (Riedel de Haen, Seele, Germany) in PBS (1 mg/ml) and the specimens were



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incubated in a dark environment for 10 min at room temperature prior to being extensively washed with distilled water and observed under an epifluorescence light microscope (Axiophot, Zeiss, Oberkochen, Germany).

Cell viability test (WST-1) The viability and proliferation of the osteoblast-like cells were quantified by means of a tetrazolium salt WST-1 assay which directly measures the cellular metabolic activity through the biodegradation of tetrazolium salt by viable and active cells. The osteoblastpopulated scaffolds (Coll/HA/PLCL and PLCL) were cultured in the presence of a 1% (v/v) WST-1 solution (5 mg/ml) for 1 h at 37 C and 5% CO2. Following this culture period, 200 l (in triplicate) of the supernatant was transferred from each scaffold to a 96-well flatbottom plate and the absorbance of the formazan dye was determined with a microplate spectrophotometer at a wavelength of 420 nm (xMark).

Chemical analyses Following a series of culture periods, we examined the morphology and chemical composition of the minerals deposited in the osteoblast-seeded scaffolds under a JEOL 5300 SEM equipped with an energy dispersive spectrometer (EDS). The scaffolds were sputter-coated with gold using a sputter coater (JFC-1200 Fine Coater, JEOL, Tokyo, Japan) and subsequently analysed and photographed at various magnifications. The minerals or bone nodules were analysed by means of the EDS and the spectra were recorded. This was supported by FTIR analyses. Indeed, the infrared spectra of the nodules were collected with a Nicolet Magna 550 Fourier transform infrared spectrometer (Thermo-Nicolet, Madison, WI). A total of 150 scans were recorded at a resolution of 4 cm1 and OMNIC (Nicolet Co.) software was used for data acquisition and spectrum processing.

Micro-computed tomography (Micro-CT) analysis Mineralised and as-prepared scaffolds were analysed by means of a SkyScan Micro-CT (model 1172, Kontich, Belgium) at a rotation step of 0.3 and an image resolution of 4.92 mp. Reconstruction was performed with the NRecone version 1.6.3.2 (SkyScan) and an analysis was performed with the CTAn version 1.11.4.2 (SkyScan). A 3D volume rendering was done with the CTvox version 2.1.1 (SkyScan). Also analysed were parameters such as porosity, object connectivity and object size before and after cell culture.

Statistical analysis For each analysis, the values were reported as the means SD based on six experiments. To test the significance of the observed difference between the experimented groups, an unpaired student t-test (except otherwise stated) was applied, with a value of p < 0.05 considered to be statistically significant.

Results Human DPSC differentiation into osteoblasts Following extraction, culture and phenotyping, all of the DPSCs exhibited the expression of CD73, CD90 and CD105, with a low expression of CD34, CD45, CD11b, CD19 and HLA-DR, confirming the stem cell phenotype of the pulp extracted stem cells (data not shown). These DPSCs were cultured in the presence or absence of osteoblast differentiating media for three weeks, after which time the osteoblast-like cells derived from the DPSCs displayed significant ARS staining (an indicator of calcium deposition) which was two folds higher in the osteoblast-inducing medium than in the normal medium (Figure 1a). In addition, the alkaline phosphatase activity showed significantly higher absorbance levels in the DPSCs differentiated into osteoblast cultures than in the normal DPSC culture (Figure 1b). Finally, Runx-2 gene expression revealed an mRNA expression that was 40% higher in the osteoblast cultures than in the DPSC culture (Figure 1c). Our results thus confirm the differentiation of the human DPSCs into osteoblastic phenotype.

Morphology of the osteoblast-like cells The SEM photomicrographs in Figure 2 show that after 8 h of culture, a significant density of osteoblastlike cells had adhered to the Coll/HA/PLCL scaffold (arrow). These cells presented long, fine pseudopodia, indicating a good adhesion and contact with the Coll/ HA/PLCL scaffold surface. In contrast, the cells on the PLCL scaffold appeared flattened and presented no evident cytoplasmic extensions. At 24 h, more adherent cells were found on the surface of the Coll/HA/PLCL scaffold than on the surface of the PLCL scaffold.

Adhesion and viability of the osteoblast-like cells Hoechst staining (Figure 3a) revealed that the osteoblast-like cells cultured in the Coll/HA/PLCL and PLCL scaffolds for 8 h displayed well-rounded nuclei that were slightly stained by the Hoechst dye, with no sign of apoptosis, such as chromatin condensation or fragmentation. The cells had adhered over the entire



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Journal of Biomaterials Applications 0(0) (a) ARS staining and CPC extraction/104 viable cells

1

Cell proliferation into the Coll/HA/PLCL scaffold and express osteogenic genes

P < 0.001

0.8

Figure 4 shows the growth of the osteoblast-like cells following culture on the Coll/HA/PLCL and PLCL scaffolds. The proliferative rate of the osteoblasts in the Coll/HA/PLCL scaffold was found to be more than twice that of the osteoblasts cultured in the PLCL scaffold. Cell proliferation became stabilised or dropped slightly at day 21, probably due to the confluence of cell growth. A significant difference was found between the two types of scaffolds at all of the time points under study. This was further supported by the osteoblast gene activation (Figure 5), as Runx2, Osterix, osteocalcin and RUNKL genes were highly expressed by osteoblast-like cells cultured into the Coll/HA/PLCL scaffold as compared to PLCL scaffold. These results suggest that the Coll/HA/PLCL scaffold promoted osteoblast growth and differentiation.

0.6 0.4 0.2

(b)

ALP activity/104 viable cells

0

RUNX-2 gene expression (Normalized fold)

(c)

0.35

P < 0.001

0.3 0.25 0.2 0.15 0.1 0.05 0 2.8

Alkaline phosphatase activity

P < 0.001

2.4 2 1.6 1.2 0.8 0.4 0 Osteogenic medium

Normal medium

Figure 1. Human DPSC differentiation into osteoblast-like cells. (a) ARS staining and CPC extraction; (b) Alkaline phosphatase activity; (c) RUNX-2 gene expression by qRT-PCR.

surface of the scaffold surface and had infiltrated pores. At 24 h, the cells covered both the surface and the bottom of the Coll/HA/PLCL scaffold, which indicates a good infiltration of the cells into the 3D matrix. Figure 3(b) shows the cell viability as determined by the WST-1 assay. Eight hours post-culture, the optical density (proportional to the number of viable cells) was approximately 0.5 for the PLCL scaffold and 0.8 for the Coll/HA/PLCL scaffold, confirming the adhesion of the osteoblast-like cells from both scaffolds and their greater affinity for the Coll/HA/PLCL scaffold. At 24 h post-culture, the optical density had increased to 1.1 and 1.5 for the PLCL and Coll/HA/PLCL scaffolds, respectively. Overall data show that the Coll/HA/ PLCL composite scaffold promoted osteoblast adhesion and viability.

Figure 6 shows the ALP activity of the osteoblast-like cells cultured on the Coll/HA/PLCL and PLCL scaffolds. ALP activity on both scaffolds increased with culture period. Moreover, ALP activity on the Coll/ HA/PLCL scaffold was found to be approximately two folds higher than that on the PLCL scaffold. A significant difference was found between the two types of scaffolds at all of the time points under study.

Calcium deposition Calcium deposition was investigated by means of the CPC-extracted ARS stain. As shown in Figure 7, a general observation was that calcium deposition on the Coll/HA/PLCL and PLCL scaffolds significantly increased from day 4 to day 21 in a time-dependent manner. Indeed, the optical density of the CPC increased from 0.6 at day 4 to 2.5 at day 21 in the PLCL scaffold. In the Coll/HA/PLCL scaffold, on the other hand, the CPC absorbance increased from 1.2 at day 4 to 6.8 at day 21, with roughly double the results.

Bone nodule morphology and composition Figure 8(a) and (b) show the SEM photomicrographs of the mineral nodules formed on the scaffolds at different time periods. The squares drawn on the SEM photomicrographs indicate the centre of the X-ray microanalysis (EDS). Beginning at day 4, cauliflowerlike mineral nodules of approximately 1 mm in diameter were observed on the Coll/HA/PLCL scaffold and became more frequent and larger to sometimes cover



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24 hours

8 hours

Coll/HA/PLCL scaffold

Figure 2. Scanning electron micrographs of osteoblast-like cells cultured on Coll/HA/PLCL and PLCL 3D scaffolds for 8 and 24 h.

a large area of scaffold surface (Figure 8a: seven days). The qualitative elemental analysis with EDS revealed that these mineral nodules were mainly composed of calcium and phosphate, the primary constituents of mineralised bone matrix. The mineralisation rate at all of the culture periods was higher in the Coll/HA/ PLCL scaffolds than in the PLCL control samples.

Bone nodule chemical analysis The chemical structure of the bone nodules was assessed by FTIR analysis in the region of 400–1800 cm1 (Figure 9a), with the band assignment summarised in Table 2. The bands at 1650, 1550 and 1270 cm1 were attributed respectively to the amide I (C ¼ O stretching of proteins), amide II (C–N stretching and N–H bending of proteins) and amide III regions of the Coll. The bands at 1425 cm1 were assigned to the 3(CO32) mode of the carbonate in the HA. The bands at 1070 and 1040 cm1 were assigned to asymmetric absorptions of the phosphate groups 3(PO43) in the HA, and the band at 1130 cm1 corresponded to the absorption of 3(HPO43), which was the non-stoichiometric hydroxyapatite (newly formed HA). Thus our results show that the osteoblast-like cells displayed the ability to produce new mineralised bone matrix when cultured on the Coll/HA/PLCL 3D scaffold.

COLL/HA/PLCL composite scaffold microstructure Figure 9(b) shows two reconstructed 3D structures of 0.5 (deep, from surface, or thickness in the middle) 0.5 (wide) 1.0 (100 cuts, 5 mm thick per cut) mm3 in volume. This figure also shows the open connected pores of the designed Coll/HA/PLCL scaffold and the clearly increased density of the scaffold following two weeks of culture with the DPSC-derived osteoblast-like cells. A 3D quantitative analysis was performed in a volume of 0.25 0.5 0.5 mm3 (100cuts) for the Coll/HA/PLCL scaffold and a volume of 0.25 0.5 0.25 mm3 (50 cuts) for the PLCL scaffold, both at the surface and in the middle of the specimens (Table 3). The porosity, total number of objects (solid matter) and Euler connectivity (connections of an object to the surrounding objects) of the Coll/HA/PLCL scaffold dramatically decreased after two weeks of culture with the osteoblasts. These data reveal the increased volume of the total solid matter and include the growing size of the solid objects in the scaffold. The decreased connectivity signifies the merging of the originally multiple small connections among objects. It is also clear that most of the pores were open pores, as the open porosity matched well with the total porosity. The PLCL scaffolds displayed a similar change but to a significantly lesser extent.



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Coll/HA/PLCL

24 h

8h

(a)

1.8

DPSC adhesion to the Scaffolds (Absorbance at 550 nm)

1.4

8h 24 h

P