Preparation of biomaterials
Demineralized bone matrix was prepared from bone allografts as previously described [12]. Concisely, the allograft bone was cleaned with 3% hydrogen peroxide and 70% isopropanol. The washed ground bone was pulverized into small particles, and the minerals were dissolved by an acid extraction process. Pulverized bone of less than 1000 μm particle size was selected for the next process. The bone was demineralized in 0.1 M HCl for 8 h with continuous stirring at 16 °C. The resulting powder was washed, lyophilized, and sieved into a particle size range of 250–500 μm. A gelatin (G) sample prepared by acidic treatment of porcine skin collagen (isoelectric point = 9) was kindly supplied by Nitta Gelatin, Osaka, Japan. Alaska crab water-soluble chitooligosaccharide (COS, MW = 2000 Da, deacetylation degree = 99%) was obtained from Hangzhou Garden Corporation, China. Other chemicals used were analytical and cell culture-tested grades.
Fabrication of three-dimensional scaffolds
Three-dimensional scaffolds of G, G/COS (GC), and G/COS/DBM (GCD) were fabricated via freeze-drying and chemical crosslinking techniques (Fig. 1). Briefly, 2 wt% aqueous pure gelatin solution was prepared in 1% (v/v) acetic acid solution (pH 2.5) for 2 h at 60 °C. Next, the other blending ratio of gelatin to chitooligosaccharide (G/COS) 70/30 for 2 wt% aqueous solution was prepared in the same solvent and conditions. The dissolved solutions were added with 0.15% (v/v) glutaraldehyde solution and stirred for 15 min. The solutions were crosslinked under 4 °C for 12 h. The gelatin blended chitooligosaccharide (GC) solution 70/30 was added with demineralized bone powder in the blending ratio of 60/40 (GCD) and then mixed until homogeneous. All of the solutions were loaded into sterile 48-well culture plates and gradually frozen at -20 °C and -80 °C for 12 h. The freeze solution was freeze-dried using lyophilizer under vacuum for 24 h. The obtained scaffolds were immersed in 0.1 M glycine solution at room temperature for 2 h to block the non-reacted aldehyde groups and repeatedly washed with sterile deionized water before refreeze-drying. The scaffolds were sterilized before cultivation using ethylene oxide.
Scanning electron microscopy
Scaffolds were cut horizontally with razor blades and sputter-coated with gold (Ion sputtering device, JFC 1100, Japan). Cross-sectional features of scaffolds were visualized by a scanning electron microscope (SEM, Jeol JSM 5400, JEOL, Japan) at an accelerating voltage of 12–15 kV after sputter-coating with gold. The pore diameter of the scaffold was analyzed from SEM micrographs via ImageJ Launcher program (n = 100).
Porosity
Pore size of each scaffold was randomly analyzed from 100 pores/scaffold using ImageJ software (the US National Institutes of Health, USA). The porosity of all prepared samples was assessed by a liquid displacement method as described previously [13]. Hexane was used as the displacement liquid as it permeates through the scaffolds without swelling or shrinking the matrix. A known weight of dry scaffold was completely immersed in a known volume (V1) of hexane for 5 min. The volume of hexane and the hexane-impregnated scaffolds were recorded (V2). The hexane-impregnated scaffold was then removed and the residual volume of hexane was recorded (V3). Porosity was calculated using the following equation: scaffold porosity (%) = [(V1 − V3)/(V2 − V3)] × 100.
Swelling ratio of the scaffolds
The initial dry scaffolds were weighed and recorded (WD). Swelling properties of the scaffolds were determined by their immersion in phosphate buffered saline (PBS, pH 7.4) for 24 h at the physiological temperature (37 °C). After the predetermined time period, the swollen scaffolds were removed from the medium and weighed (WS). The swelling ratio was calculated according to the following equation: swelling ratio = (WS – WD)/WD.
Compressive strength
A universal mechanical tester (Instron 5567, USA) was used to evaluate compressive modulus of scaffolds by compressing the scaffolds (13 mm in diameter and 3 mm in thickness) at a constant rate of 0.5 mm/min. The end point of compression was set at 50% of initial thickness of the scaffolds. The slopes of compressive stress–strain curves at 5–35% deformation were used to calculate the compressive modulus of the scaffolds.
Isolation and culture of mesenchymal stem cells (MSCs)
The MSCs were isolated from human periosteum using primary outgrowth techniques [14]. The cells were incubated for 30 min at 4 °C with monoclonal antibodies against human anti-CD29, CD34, CD44, CD45, CD90, and CD105 conjugated with PE, FITC, APC, and PE/Cy5 (BioLegend, USA). Stained samples were analyzed using a flow cytometry (BD Biosciences, San Jose, CA, USA). As shown in a previous report [14], the periosteum-derived primary cells expressed MSC specific markers (CD29, CD44, CD90, CD105), while they did not express hematopoietic stem cell surface markers (CD34, CD45). Additionally, they exhibited the tri-lineage capacity to differentiate into osteogenic, chondrogenic, and adipogenic lineages and expressed marker genes for osteoblasts, adipocytes, and chondrocytes. The MSCs were cultured in alpha-minimum essential medium (α-MEM, Hyclone, Logan, UT, USA) supplemented with 10% fetal bovine serum (FBS, Hyclone, Logan, UT, USA) and 100 unit/ml penicillin/100 μg/ml streptomycin and incubated in a 5% CO2 incubator at 37 °C.
Initial attachment and proliferation assay
MSCs (5.0 × 105 cells/scaffold) were seeded onto the ethylene oxide-sterilized scaffolds and cultured in proliferating medium (α-MEM supplemented with 15% FBS) at 37 °C in a 5% CO2 incubator. The number of cells attached on the test scaffolds after 6 h seeding and cells proliferated on days 1, 3, 5, and 7 was determined using fluorometric quantification of cellular DNA according to the method reported by Takahashi [15]. Briefly, the cell-seeded scaffold samples were lysed in saline sodium citrate buffer (SSC, pH 7.4) containing sodium dodecyl sulfate at 37 °C overnight. Then, 100 μl of cell lysates were mixed with a fluorescent dye solution (Hoechst 33258 dye) in a 96-well black plate. Fluorescence intensities of the mixed solutions were spontaneously measured at the excitation and emission wavelengths of 355 and 460 nm, respectively. The standard curve between the DNA and cell number was prepared using standards of known cell numbers. The DNA assay was conducted three times independently for every experimental sample unless otherwise mentioned.
Osteogenic differentiation of MSCs on scaffolds
MSCs (1.0 × 106 cells/scaffold) were seeded on the scaffolds in proliferating medium under continuous shaking for 6 h. After seeding for 1 day, the medium was changed to osteogenic medium (α-MEM supplemented with 10% FBS, 50 μg/ml L-ascorbic acid, 10 nM dexamethasone, and 10 mM β-glycerol phosphate) and changed 3 times a week. The experiments were performed for 4 weeks in osteogenic medium. Osteogenic differentiation markers, including alkaline phosphatase (ALP) activity and calcium release were determined by p-nitrophenyl phosphate and O-cresolphythalein methods, respectively. The number of cells determined by the fluorometric quantification of cellular DNA was used to normalize the ALP activities and calcium contents.
Elemental analysis of cell surface cultured scaffolds
Elements, especially calcium (Ca), phosphorous (P), and oxygen (O) on the cell surface after 28-day culture in osteogenic media were analyzed by energy-dispersive X-ray spectroscopy (EDX, Philips Model XP 30 CP, USA). The same cell-seeded constructs used in SEM observations were used for EDX analysis.
Osteogenic potential of scaffolds in in vivo bioassay
All procedures followed home office guidelines on the scientific use of animals (Scientific Procedures) Act 1986. All animal experiments were conducted in agreement with the Chulalongkorn University Animal Care and Use Committee and with ethical approval from the Institutional Review Board of the Faculty of Medicine, Chulalongkorn University.
Eight-week-old male Wistar rats (275–300 g) were used in this study. The G, GC, and GCD scaffolds (2x11x11 mm3) were implanted into the subcutaneous tissue to evaluate the formation of ectopic bone tissue under standard sterile conditions. For each rat, the samples and control were placed into the left and right sides of the back. Gelfoam, a medical device used as hemostatic material for bleeding surfaces, served as a control. The rats were anesthetized by intraperitoneal injection of thiopental sodium (60 mg/kg body weight). The rats were shaved and disinfected with betadine solution and then 70 vol% ethanol. A vertical incision was created down the midline of the back. A 1 cm skin incision was created to form four pockets in the subcutaneous tissue. The scaffolds were inserted subcutaneously away from the incision. The wound was sutured with 4–0 prolene suture and cleaned with betadine solution.
Histological examination
A total of 48 scaffolds were implanted into 12 rats (4 scaffolds per rat). The rats were divided into 2 groups (4 and 8 weeks). After 4 and 8 weeks of implantation, the rats were sacrificed with an overdose of thiopental sodium. The scaffold implants were histologically analyzed for the formation of ectopic bone tissue by hematoxylin and eosin (H&E) and von Kossa staining. The scaffold samples and surrounding tissues were retrieved, fixed in 10 vol% formalin, and then embedded in a paraffin block. The paraffin-embedded tissues were cut (5 μm thick sections) and followed by staining with H&E to observe collagen formation, cell infiltration, and remaining scaffold. The newly formed collagen and calcium deposition induced by the implant scaffolds was perceived using von Kossa staining. Concisely, the cross-sections of scaffold samples were stained with 5 wt% silver nitrate solution, followed by washing with 5 wt% sodium thiosulphate and double-distilled deionized water. The stained sections were histologically observed and images were taken using an Olympus AX80 Provis microscope (Olympus Ltd., Tokyo, Japan). The experiment was independently duplicated. Each experiment was executed to obtain three samples for each experimental group (n = 3).
Statistical analysis
All statistical analyses were performed using the Statistical Package for Social Sciences (SPSS), version 17.0 (SPSS Inc., Chicago, IL, USA). All the experiments were carried out at least three times with triple replications. Data were expressed as mean ± standard deviation (SD). Differences between the experimental groups were examined using the one-way analysis of variance (ANOVA), with Tukey’s post hoc test if ANOVA showed significance. P-values < 0.05 were considered statistically significant.