In Vitro Biocompatibility and Osteoblast Differentiation of an Injectable Chitosan/Nano-Hydroxyapatite/Collagen Scaffold

1 Department of Ultrasonic Diagnosis, Zhujiang Hospital of Southern Medical University, Guangzhou 510282, China 2 School of Geosciences and Info-Physics, Central South University, Changsha 410083, China 3 Key Laboratory for Biomechanics and Mechanobiology of Ministry of Education, School of Biological Science and Medical Engineering, Beihang University, Beijing 100191, China 4 Department of Orthopedics, Zhujiang Hospital of Southern Medical University, Guangzhou 510282, China 5 State Key Laboratory of New Ceramic and Fine Processing, Tsinghua University, Beijing 100084, China


Introduction
Annually, more than 2.2 million bone grafting procedures (autologous bone graft and banked bone) are performed worldwide to ensure adequate bone healing in many skeletal problems, such as nonunion fractures, cervical and lumbar spine fusion, joint arthrodesis, and revision arthroplasty [1]. Tissue engineering offers a strategy to circumvent those problems. The concept involves the use of a porous and biodegradable scaffold, allowing cells to adhere and proliferate, creating conditions for the formation of ECMlike structures [2][3][4]. Previous studies have shown that natural-based polymers such as chitosan [5][6][7][8][9][10] have great potential for bone tissue engineering applications. The main advantages of these materials include low immunogenic potential, bioactive behavior, good interaction with host tissues, chemical versatility, and high availability in nature. Chitosan is a promising material for bone regeneration because it is biocompatible and biodegradable with a degradation rate that is dependent on factors such as degree of deacetylation (DDA) and crystallinity, and it can be easily formed into beads, fibers, or more complex structures [5,11,12].
Bone tissues are mainly constructed from nanosized hydroxyapatite (HA) minerals and type 1 collagen (Col-1) matrix with complex hierarchically assembled structures [13][14][15][16]. The ideal scaffolds for bone regeneration should promote early mineralization and support new bone formation [17][18][19]. Mineralized collagen fibrils (nHAC) composites seem to be very promising biomaterials for bone regeneration [20][21][22][23]. In the previouse study, the feasibility of developing a thermosensitive and injectable chitosan 2 Journal of Nanomaterials solution in the presence of nHAC was demonstrated [23][24][25]. Combining nHAC and chitosan has the potential to maximize the beneficial properties of each and creates an injectable scaffold with properties similar to physiological bone which would undoubtedly aid in the formation of new bone at the tissue/biomaterial interface [24][25][26][27].
In the present study, the cytocompatibility of the injectable CS/nHAC scaffolds to act as a bone substitude and its potential for bone tissue engineering were investigated in vitro. The ability of the injectable CS/nHAC scaffolds to maintain the viability and functionality of seeded MC3T3-E1 preosteoblasts was investigated in terms of their proliferation and osteoblastic differentiation over 10 days of culture.

Scaffold
Processing. The CS/nHAC composite was prepared by the procedure reported previously [24][25][26][27]. nHAC powder was synthesized by self-assembly of nanofibrils of mineralized collagen and sterilized by γ-ray irradiation (1.5 Mrad). Chitosan (2 g) was dissolved in hydrochloric acid solution (98 mL, 0.1 M). The nHAC powder was added to the chitosan solution (0.02 g/mL). Finally, the pH of the CS/nHAC solution was adjusted to 7.0 by adding droplets of β-glycerophosphate solution (30% (w/v)). The samples for biological tests were prepared in line with the aseptic technique in an aseptic manipulation cabinet. Solutions were injected into a circular mold (12 mm diameter and 2 mm thickness) and solidified in an incubator at 37 • C for 10 min to form a hydrogel scaffold.
The CS was prepared similar to the procedure of CS/nHAC. Chitosan (2 g) was dissolved in hydrochloric acid solution (98 mL, 0.1 M), and then the pH of the CS solution was adjusted to 7.0 by adding droplets of β-glycerophosphate solution (30% (w/v)). The samples for biological tests were prepared in line with the aseptic technique in an aseptic manipulation cabinet.

Cell Culture
Studies. MC3T3-E1 murine calvarial osteoblasts (subclone 14) cells were purchased from the cell bank of the Chinese Academy of Sciences (Shanghai, China). The cells were grown in a culture medium consisting of α-MEM (Sigma, St. Louis, MO), 10% fetal bovine serum (Biochrom AG, Germany), and 1% of antibioticantimycotic mixture (Sigma, St. Louis, MO) at 37.8 • C in a humidified atmosphere of 5% CO 2 . When an adequate cell number was obtained, cells at passage 2 were detached with trypsin/EDTA. Cells were seeded at a density of 2 × 10 4 cells/scaffold under static conditions, by means of a cell suspension. The culture medium was changed every 2-3 days until the end of the experiment.

In Vitro Cytotoxicity Tests.
A Cell Count Kit-8 (Cell Count Kit-8, Beyotime, China) was employed in this experiment to quantitatively evaluate the cytotoxicity of the scaffold. Cell viability was assessed after MC3T3-E1 were inoculated on the samples 24-well disks using the CCK-8 kit after 1, 3, 7 and 10 days. CCK-8 reagent (100 μL) was added to MC3T3 in 1 mL medium per well. The plates were incubated at 37 • C for 4 h and shaken for 1 min. After this, 200 μL (n = 5) were transferred to 96-well plates and the optical density (OD) was measured on a microplate reader (BioTek, USA) using an absorbance of 490 nm. All the procedures were replicated 3 times.

Cell Proliferation by DNA Quantification.
MC3T3 proliferation on the CS/nHAC scaffolds was determined using a fluorimetric dsDNA quantification kit (PicoGreen, Molecular Probes, Invitrogen, USA). Samples collected at days 1, 3, 7, and 10 were washed twice with a sterile phosphatebuffered saline solution and transferred into 1.5 mL microtubes containing 1 mL of ultrapure water. Cell constructs were cryopreserved at −80 • C for further analysis. Prior to DNA quantification, samples were thawed and sonicated for 15 min. Standards were prepared with concentrations ranging between 0 and 2 mg/mL. Per each well of an opaque 96-well plate were added 28.7 μL of sample (n = 3) or standard, 71.3 μL of PicoGreen solution, and 100 μL of Tris-EDTA buffer. The plate was incubated for 10 min in the dark and fluorescence was measured using an excitation wavelength of 260 nm and an emission wavelength of 280 nm. All the procedures were replicated 3 times.

Alkaline Phosphatase Quantification.
After culturing for 1, 3, 7, and 10 days, the cells were washed and lysed in 0.1 vol% Triton X-100. The alkaline phosphatase (ALP) activity in the lysis was determined through a p-nitrophenyl phosphate (pNPP) liquid substrate system (Nanjing Jiancheng, China). Five milliliters of each cell lysate solution was added to 195 μL of pNPP substrate and incubated in the dark at room temperature for 1 min. The absorbance was read using a plate reader (Molecular Devices, USA) at 405 nm. The intracellular total protein content was determined using a Micro BCA Protein Assay Kit (Nanjing Kaiji, China) and the ALP activity was normalized to it.

Osteogenic Differentiation by Reverse Transcriptase PCR.
The expression levels of osteogenesis-related genes were measured using the quantitative reverse transcriptionpolymerase chain reaction (qRT-PCR). The cells were seeded at a density of 2 × 10 4 cells/well, cultured for 1, 3, 7 and 10 days, and then harvested using TRIzol (Gibco) to extract RNA. An equivalent amount of RNA from each sample was reverse-transcribed into complementary DNA (cDNA) using the Superscript II first-strand cDNA synthesis kit (Invitrogen). The qRT-PCR analysis of genes including type 1 collagen (COL-1), osteocalcin (OCN), Runt-related transcription factor 2 (RUNX-2), and 18S ribosomal RNA (18 s rRNA) was performed on the Applied Biosystems 7500 using the Quantitect Sybr Green Kit (Qiagen). The primers for the target genes were listed in Table 1. The Ct values of target genes were normalized by the Ct values of the TaqMan

Statistical
Analysis. Statistical significance of differences was determined using one-way and two-way analysis of variance ANOVA. If differences were detected, pairwise comparisons were made using Tukey's HSD test at a confidence interval of 95% (P < 0.05).

In Vitro Cytotoxicity
Tests. The viable cell density increases over initial days up to a plateau based on the CCK-8 assay (Figure 1). The experimental group and control group share similar trend of viable cell density over 10 days. the CS/nHAC group reach a maximum value at day 10, and the CS group reach a maximum value at day 7; no significant growth difference was observed between the two groups after 10 days of incubation (n = 5, P > 0.05). The results of the CCK-8 assay reflect an overall activity which is affected by both cell proliferation and cell viability. The cell proliferation assay showed that cells grew normally with the scaffolds and these scaffolds were nontoxic for MC3T3-E1.

Cell Proliferation by DNA Quantification.
Over the 10 days of the study, the number of cells, as measured by dsDNA, was essentially equivalent between both CS and CS/nHAC scaffold groups ( Figure 2). Overall, there was not a significant amount of cell proliferation detected over the course of the study. In vitro cell testing of biomaterials is a well-established method to determine the cytocompatibility of the materials [27][28][29] by using either cell lines or primary cells. This study was designed primarily to analyze the ability of the composite scaffold to support osteoblast matrix production, and maintaining similar cell numbers in each group allowed differences in these parameters to be studied without the confounding effects of cell number differences. The DNA concentration result of the two groups showed a similar trend to the CCK-8 result. These results demonstrated that both the CS and CS/nHAC scaffold had good cytocompatibility and MC3T3-E1 viability, so it highlights the application of CS/nHAC to be a biomaterial for bone tissue engineering.

Alkaline Phosphatase
Quantification. ALP activity and calcium mineralization are mostly used as markers for early and late differentiation of osteoblast cells, respectively [21,22]. Osteoblastic cell differentiation was assessed by measuring ALP activity normalized to total protein content after 1, 3, 7, and 10 days of culture. Figure 3 shows the * * * * evolution of ALP activity for MC3T3-E1 cells cultured on the CS/nHAC surfaces in comparison with CS. The ALP activity of MC3T3-E1 cells grown on both groups was not significantly different for day 1 and 3. However, MC3T3-E1 cells cultured on CS/nHAC scaffold had significantly higher ALP activity than those cultured on CS group on day 7 and 10 (P < 0.001). The ALP activity of CS/nHAC group reaching a top on day 7 was observed. The following decrease on day 10 is likely due to reaching an advanced cell culture stage. ALP activity is mostly used as markers for early and late differentiation of osteoblast cells, respectively [30,31]. These results indicate that the CS/nHAC scaffold can stimulate matrix formation and enhance osteoblast cell differentiation.

Osteogenic Differentiation by Reverse Transcriptase PCR.
A semiquantitative RT-PCR method was used to assess gene expression of osteoblasts after 1, 3, 7, and 10 days. In order to know whether primary osteoblast differentiation was affected by nano-hydroxyapatite/collegen, we selected COL-1, OCN, and RUNX-2 as markers for osteoblastic differentiation in this study. Among bone matrices, COL-1 is the most abundant protein synthesized by active osteoblasts and conductive to mineral deposition. Osteocalcin is secreted solely by osteoblasts and thought to play a role in the body's metabolic regulation and is proosteoblastic or bone-building [28]. It is also involved in bone mineralization and calcium ion homeostasis. RUNX-2 gene is a member of the RUNX family of transcription factors and encodes a nuclear protein with a Runt DNA-binding domain. The protein can bind DNA both as a monomer and, with more affinity, as a subunit of a heterodimeric complex. This protein has been implicated as a key transcription factor associated with osteoblast differentiation [29]. The results of real-time PCR for mRNA expression of osteocalcin and RUNX-2, both of which are osteoblast markers, are represented as fold increases. As shown in Figures 4(a) and 4(b), there are significant differences in the osteocalcin expression between CS/nHAC and CS at day 1, 7, and 10 (P < 0.001) (Figure 4(a)). The osteocalcin expression was 5.5-fold higher on day 7 and 2.7fold higher on day 10 for the CS/nHAC as opposed to the CS. Also, the RUNX-2 expression was significantly higher by day 7 and 10 for the CS/nHAC as opposed to the CS group. The COL-1 expression was significantly lower by day 1 and 3 for the CS/nHAC as opposed to the CS group. However, significant higher COL-1 expression of CS/nHAC than CS was observed at day 7 and 10 (P < 0.01). Due to the current results in vitro, better osteoblasts viability, and differentiation marked by higher OCN, RUNX-2 and COL-1 expression were observed on CS/nHAC surfaces. The addition of nHAC to the chitosan did lead to significantly enhanced osteoblasts differentiation. There are several factors that could contribute to the enhanced mineralized matrix production seen when osteoblasts were cultured on CS/nHAC scaffolds. When calcium phosphatecontaining materials are hydrated in cell culture media, a continuous process of calcium and phosphate ion dissolution and reprecipitation occurs. This creates a dense carbonated apatite layer on the surface of the scaffold that is similar to the structure of native bone and may therefore induce more rapid osteoblast differentiation and mineralization [30][31][32]. Taken together, these gene expression data associated with the osteoblast phenotype provides evidence that the scaffold constitutes a good substrate for MC3T3-E1 cell differentiation leading to ECM mineralization.

Conclusions
In this study, we conclude that the CS/nHAC scaffold has a suitable biocompatibility for its use as cell culture scaffold for hard tissue regeneration. In vitro experiments revealed more prominent activation of osteoblast differentiation in cells grown on CS/nHAC than in those grown on CS. Collectively, these data indicates the CS/nHAC may give greater results concerning cell proliferation and differentiation compared to chitosan in this in vitro study. We suggest an excellent applicability of the CS/nHAC as bone substitutes.

Authors' Contribution
Y. Chen and Z. Huang contributed equally to this paper.