Original
Synthesis of a Novel bFGF/nHAP/COL Bone Tissue Engineering Scaff old for Mandibular
Defect Regeneration in a Rabbit Model
Yue Cai1) , Xuexin Tan1) , Li Zhao2) , Ran Zhang1) , Tong Zhu1) , Yang Du3)
and Xukai Wang1) 1)
Department of Oral and Maxillofacial Surgery, School of Stomatology, China Medical University, Liaoning Institute of Dental Research, Shenyang, China
2) The affi liated Zhongshan Hospital Dalian University, Dalian, China
3) Department of Oral Medicine, School of Stomatology, Jinzhou Medical University, LiaoNing Province, China
(Accepted for publication, October 24, 2017)
Abstract: In bone tissue engineering, scaffold fabrication and biocompatibility are crucial concerns. Many scaffold materials have been explored, among which nanometer hydroxyapatite (nHAP) and collagen (COL) are commonly used. Additionally, growth factors can be used to modify scaff olds. In this study, lyophilization technology was used to build a scaff old comprising basic fi broblast growth factor (bFGF), nHAP and COL for the fi rst time. The resulting scaff old was characterized. bFGF release from the scaff old was assessed by ELISA. Bone marrow mesenchymal stem cells (BMSCs) were prepared and seeded onto the scaff old to test in vitro biological compatibility. A scanning electron microscope was used to observe the scaff old and evaluate BMSC morphology, and the cells were counted to detect early cell adhesion. Cell proliferation and activity were assessed by a cell counting kit-8 assay and measurement of alkaline phosphatase activity, respectively. Bilateral mandibular defects were prepared in 12 New Zealand rabbits and repaired using scaff olds. The rabbits were divided into four groups: a group treated with allogeneic BMSC-seeded bFGF/nHAP/COL scaff old, a group treated with allogeneic BMSC-seeded nHAP/COL scaff old, a group treated with nHAP/COL scaff old alone, and an untreated control group. After 12 weeks, three-dimensional computerized tomography examination, computerized tomography value measurement, gross observation and hematoxylin and eosin stain staining were conducted. SPSS17.0 software was used for data analysis. The gross morphology conformed to the characteristics of a tissue engineering scaff old. The bFGF/nHAP/COL scaff old promoted BMSC adhesion, proliferation and diff erentiation and hence promoted good bone formation, without exhibiting biological toxicity. Our fi ndings show that the bFGF/nHAP/COL scaff old has good physical properties and biocompatibility in vitro, and can be used to promote osteogenesis after in vivo implantation. Key words: Tissue engineering, Bone defect, nHAP, COL, bFGF
Introduction
Bone is an organ with a complex cellular composition, and bone tissue, with its specialized organic and inorganic architecture, can be defined as microscopic nanocomposite tissue1)
. Bone defect repair involves a series of complex physiological and pathological processes2).
Comminuted fractures, bone tumors, extractions and infections often result in local loss of bone tissue in varying degrees3). Bone grafts
and prosthesis implantation are used for bone defect repair and reconstruction, but the results are often unsatisfactory. The eff ectiveness of bone grafting is restricted mainly by limited donor bone supply and potential complications. Acellular allogeneic bone bypasses this limitation, but complications such as nonunion, infection, and stress fractures often occur4).
Bone tissue engineering emerged almost thirty years ago as an alternative approach to repairing bone defects. Since then, this field has received increasing attention and techniques have substantially improved5). In general, an engineered tissue consists of a scaffold,
cells, and essential growth factors6-8). Scaffolds with high porosity
promote bone regeneration and vascularization9). therefore, the
selection of materials and construction of scaff olds are very important considerations. Collagen, a biologically inducible, biodegradable substance with ideal mechanical properties is a major extracellular
matrix component that is widely used in structural reconstruction and promotes cell growth and differentiation10)
. At the same time, nano-scale biomaterials have attracted wide attention in the field because of their good biological and biomechanical properties11)
. Among various nanomaterials, nanometer hydroxyapatite (nHAP) has been investigated for use mainly because of its potential biocompatibility and osteoinductive properties. nHAP is essentially the same as natural hydroxyapatite, so it has become a common material for bone regeneration and reshaping applications, and dental restoration, partly because it can enhance cell attachment, proliferation, and mineral formation12,13).
Basic fi broblast growth factor (bFGF), a member of the fi broblast growth factor family, has a variety of biological regulatory functions, including neuroprotection, inducing vasodilation, stimulating angiogenesis and inhibiting apoptosis14-16). Wang Lei et al. previously
confirmed that bFGF (50 ng/ml) promoted BMSC proliferation17). In
the present study, lyophilization was used to mix bFGF with nHAP and collagen (COL) to form a composite scaff old (bFGF/nHAP/COL). Vilquin et al. previously elucidated that bone marrow mesenchymal stem cells (BMSCs) are ideal for use in tissue engineering because they exhibit no immunogenicity18). Early experiments demonstrated that the
critical diameter of round, full-thickness bone defects in rabbits is 8 mm19).
In this examination, a composite scaffold was constructed and seeded with cells, and was tested for biocompatibility in vivo and in
vitro to identify an ideal scaff old for bone tissue engineering.
Correspondence to: Dr. Xukai Wang, Department of Oral and Maxillofacial Surgery, School of Stomatology, China Medical University, Liaoning Institute of Dental Research, Shenyang, 117 Nan Jing North Street, Heping, Shenyang, Liaoning 110002 P.R.China; Tel: +8602422892451; E-mail: wangxukai757892@sina.com.cn
2018 The Hard Tissue Biology Network Association Printed in Japan, All rights reserved.
Materials and Methods
Materials
Rat tail tendon type I collagen was purchased from Shengyou Biotechnology (Hangzhou, Zhejiang, China). nHAP was purchased from Emperor Nano Material (Nanjing, Jiangsu, China). Acetic acid, anhydrous ethanol, hydrochloric acid (HCl), and cell counting kit-8 (CCK-kit-8) and alkaline phosphatase (ALP) kits were purchased from the Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Phosphate buffer solution (PBS), alpha minimum essential medium (αMEM), 10% fetal bovine serum (FBS), and 0.25% trypsin were purchased from GE Healthcare Life Sciences Hyclone Laboratory (South Logan, Utah, USA). bFGF was purchased from PeproTech (Rocky Hill, New Jersey, USA). bFGF ELISA kits were purchased from the Yuduo Biotechnology Company (Shanghai, China). All other general laboratory reagents were of the highest quality available and were obtained through standard commercial suppliers.
Preparation of bFGF/nHAP/COL scaff olds
Acetic acid was diluted to 0.005 mol/L in deionized water. COL (10 mg) was added to 10 ml of the acetic acid solution and stirred with a JB-2A stirrer (Bante Instruments Limited, Shanghai, China) for 50 minutes. nHAP (10 mg) was then added and the solution was stirred overnight. The quality ratio of the solution yielded by the above method (nHAP: COL) was 1:1.5. bFGF was dissolved in PeproTech protein solution to 5 ng/μl20) and then added to the scaff old solution and stirred at a low temperature for 50 minutes, so that the fi nal concentration of bFGF in the mixed solution was 50 ng/ml. The solution was then transferred to a 24-well Tefl on culture plate and frozen at −20°C for 24 h, and was then lyophilized at −80°C for 48 h using a VFD-2000 (Boyikang, China) to obtain bFGF/nHAP/COL scaff olds. The morphology and microstructure of the scaffolds were observed with a scanning electron microscope (SEM)(S-4800, Hitachi Co., Tokyo, Japan).
Determination of scaff old porosity, swelling capacity, water absorption capacity, mechanical properties and degradability
Scaffolds were cut into an appropriate shape. The volume was recorded as Vs and the quality as ms. The density was calculated as ρs = ms / Vs. Each sample was measured three times and the average value was recorded. Scaff olds were immersed into anhydrous ethanol for 24 h and then dried and weighed. The quality was recorded as m. The volume of the inner hole was recorded as Vp: Vp = (m − ms) / ρs. The porosity of scaff old was calculated as Vp / Vs. Each sample was measured three times and the average value was recorded21).
Sample volume was recorded as V1, and then each sample was immersed in deionized water at room temperature for 24 h. The wet volume was recorded as V2. The swelling capacity was calculated as (V2 / V1 − 1) × 100%22). After excess water was wiped off , the wet weight
was recorded as S1. The scaff old was dried in an oven (Yuejin Medical Optical Instruments, Shanghai, China), and the dry weight was recorded as S2. The water absorption capacity was calculated as (S2 / S1 − 1) × 100%23,24).
The compression strength and modulus were obtained by calculation of a stress–strain curve. A micro-load fatigue testing machine (MMT-101NN-10, SSM, Tokyo, Japan) was used at room temperature. The load was 100 N, and the speed was 0.5 mm/min. The diameter of each sample was 13.73 mm, and the height was 10.00 mm. Three samples were tested to obtain an average compression strength and modulus and the standard error was calculated. The compression modulus was defi ned as the slope of the initial linear part of the stress–strain curve. From 1% strain, a straight line was drawn parallel to the initial linear
section of the curve, and the ordinate of the intersection with the stress– strain curve was taken as the compression strength23)
.
The initial quality of each sample was recorded as m0, then the samples were placed in a 24-well culture plate and sterilized using a Co-60SQ(H)630 (Shanqiang Nuclear Radiation Engineering Technology, Beijing, China). The wells were fi lled with PBS, then the plates were sealed with sterile tape and placed in a cell culture incubator (Heng Scientific Instrument, Shanghai, China) at a constant temperature of 37°C. The samples were washed with deionized water and analyzed at 1, 7, 14, 28, and 56 days25). The four samples were measured, and the pH
of the degradation liquid was determined. The samples were removed from the PBS, washed and dried, and lyophilized for 48 h. The dry weight was recorded as m1. The degradation rate was calculated as (m0 − m1) / m0.
Detection of bFGF release from scaff olds
Each standard scaffold was fabricated from 1 ml bFGF/nHAP/ COL (containing 50 ng bFGF). Three scaff olds were placed into wells of a plate and 1 ml of 1× PBS buff er (pH 7.4) was added to the wells. The plates were sealed with a membrane and placed in a cell culture incubator at 37°C. PBS was collected from the pores of the samples at specific time points (1, 2 to 20 days). PBS(1 ml) was added to the collected liquid and a bFGF ELISA kit (Yuduo Biotechnology, Shanghai, China) was used to detect the bFGF content of each sample. The mean bFGF content was determined three times and cumulative bFGF release was calculated and plotted17).
Preparation and identifi cation of BMSCs
The identity of rabbit BMSCs (BioHermes, Wuxi, China) was confi rmed by inducing osteogenesis. β-glycerophosphate (10 mmol/L), dexamethasone (10 nmol/L) and ascorbic acid (50 g/ml) were added to αMEM containing 10% FBS to produce an osteogenic medium. Third-passage BMSCs were digested with trypsin. The digestion was terminated by addition of αMEM containing 10% FBS, then this mixture was centrifuged. The supernatant was discarded and the cells were resuspended in fresh medium. The cell suspension was adjusted to 2 ×105 cells/ml, and then 1 ml of cell suspension was seeded per well in
24-well plates. αMEM containing 10% FBS was added to the wells and the cells were cultured at 37°C with 5% CO2 .When the BMSCs reached
approximately 80% confl uence, the culture medium was discarded and the cells were washed three times. Osteogenic media was then added and the cells were cultured at 37 °C with 5% CO2. The culture medium
was changed every other day. On day 21 after the start of osteogenic induction, the cells were fixed with 4% paraformaldehyde. Calcium nodules were stained with 0.2% alizarin red and observed using a microscope(CKX41, Olympus, Tokyo, Japan)26).
Cell culture
The scaff olds were disinfected, then were immersed in Dulbecco’s modified Eagle’s medium containing 10% FBS for 24 h. Suspended BMSCs were adjusted to 1 × 105 cells/ml. 100 μl mMedium (100 μl)
was added into the wells of 24-well plates, and then scaffolds were placed into separate wells. The cell suspension was added to the surface and cultured for 2 h in at 100% humidity.
BMSC morphology
atmosphere. The samples were washed with PBS and dehydrated with an ethanol gradient (50%, 70%, 80%, 90% and 100%) for 20 minutes. Finally, the samples were allowed to dry naturally and were sprayed with gold for analysis with an SEM.
BMSC adhesion
The BMSCs were adjusted to 1 × 104 cells/ml and added to culture plates that were pre-coated with bFGF/nHAP/COL or nHAP/COL scaffolds (1 ml per well). BMSCs in culture plates with no scaffold served as a control group. Six wells were prepared for each group and the cells were cultured at 37°C with 5% CO2. The number of
non-adherent cells was counted at 1, 2, 4, 8, and 24 h and the adhesion rate was calculated according to the following formula: adhesion rate (%) = (number of seeded cells − non-adhered cells) / (number of seeded cells) × 10027).
CCK-8 assay
Aqueous soluble tetrazolium/formazan (CCK-8 kit, Dojindo, Maryland, USA), an indicator of NADH- and NADPH-dependent dehydrogenase activity, was used to assess cell proliferation in the two scaff old groups and the control group. At 1, 3, 5, and 7 days after incubation, three samples from each group were assessed. CCK-8 solution (100 μl) was added to each well and the culture plate was incubated at 37°C with 5% CO2 for 4 h. A 300 μl aliquot was
removed from each well and transferred to a 96-well culture plate. The absorbance of the solution at 450 nm was measured with a microplate reader (Bio-Rad, California, USA) and the average value of three samples was recorded. The optical density at 450 nm was taken as proportional to the number of cells28).
ALP activity
At 1, 4, 7 and 10 days after the start of culture, three samples were assessed from each group. The samples were washed with PBS three times, then 1 ml of 0.1% Triton X-100 was added to each well and the culture plate was placed into a freezer at 4°C overnight to disrupt the BMSC cellular structure. Then, 30 μl of cell suspension was transferred to a 96-well plate. Buff er solution (50 μl) and matrix liquid were added, and the mixture was mixed thoroughly then incubated in a water bath for 15 minutes. Next, 150 μl of chromogenic agent was added to each well and mixed by oscillation, and the optical density at 520 nm was measured by ELISA29). The total amount of protein was measured using
the bicinchoninic acid method and ALP activity was normalized to total protein. ALP activity was recorded as U/g protein.
Surgical procedure and implantation
Twelve healthy New Zealand white rabbits weighing approximately 2.5 kg were divided into four groups: a group treated with BMSC-seeded with bFGF/nHAP/COL scaffold (BMSC/bFGF/nHAP/COL group), a group treated with BMSC-seeded nHAP/COL scaffold (BMSC/nHAP/COL group), a group treated with nHAP/COL alone (nHAP/COL group), and an untreated (control) group. The rabbits were anesthetized with pentobarbital sodium (0.2 ml/kg) and bilateral 8-mm-diameter mandibular defects were created in the buccolingual direction. Eight-millimeter diameter porous scaff olds or scaff old/cell composites were inserted into the defects. All rabbits were sacrifi ced 12 weeks after implantation. The implants were excised together with the surrounding tissues, and tissue specimens were evaluated by computerized tomography (CT) and histologically.
General observation and radiographic analysis
After undergoing surgery, the rabbits were placed and fed regularly, and their general state, jaw movement, and wound healing were observed. Twelve weeks after surgery, three-dimensional (3D) images of the mandibles were captured by 3D-CT (SOMATOM Definition AS+, Siemens, Germany) and CT values were measured. After the rabbits were sacrifi ced, the mandible was removed and split from the center.
Histological analysis
Fixed tissue samples were decalcifi ed, dehydrated and embedded in paraffi n. Tissue specimens were cut into 5-μm-thick sections, stained with hematoxylin and eosin (HE; Jiancheng Biotechnology Institute) and analyzed by light microscopy (CKX41; Olympus Co., Tokyo, Japan) to detect new bone.
Statistical analysis
All data are expressed as means ± standard deviation. Analysis of variance and the Student’s t-test were used to compare groups. Data analysis was conducted using SPSS 17 statistical software (SPSS, Inc., Chicago, USA). Diff erences were considered signifi cant when P < 0.05.
Results
bFGF/nHAP/COL scaffold morphology and scanning electron microscopy
There was no apparent diff erence in morphology between the nHAP/ COL and bFGF/nHAP/COL scaffolds, so only the latter is described here. The scaffold was white and exhibited good flexibility, and gradually returned to its original state after compression deformation. The surface was slightly rough (Fig. 1). The three-dimensional structure of the scaff old was porous, and a large number of nHAP particulates Figure 1. Gross morphology of the bFGF/nHAP/
COL scaff olds.
Table 1. Porosity, pore size, swelling index, water absorption of the bFGF/nHAP/COL scaff olds. Porosity (%) Pore size (μm) Water adsorption (%) Swelling index (%) 93.1±6.0 130±29 245±5 33.4±0.9 Results are expressed as the means±SD(n=3).
Table 2. Compressive strength and compressive modulus of the bFGF/nHAP/COL scaff olds. Compressive strength (MPa) Compressive modulus (MPa) 4.24±0.92 0.58±0.12 Results are expressed as the means±SD (n=3).
8.0 7.5 7.0 6.5 1 2 3 4 5 6 7 8 9 10 11 12 1h 1d 3d 7d 10d 14d 21d 40 30 20 10 0 A B
Figure 3. (A) In the process of degradation, the PH values of bFGF/nHAP/COL scaffold were gradually increased and finally stabilized between 7.3 and 7.4. (B) The changes of degradation rate during the course of degradation. With the degradation time increased, the degradation rate of the bFGF/nHAP/COL scaff olds increased steadily.
PH Value Changes During Scaff old Degradation
D eg ra da ti on r at e (% ) PH V al ue
Time Degradation time (week)
100 80 60 40 20 0 bF G F r el ea se o f po si ti ve c on tr ol ( % ) 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 Time(days) bFGF release rate
Figure 4. Cumulative release rate of bFGF per time point from bFGF/ nHAP/COL scaff olds.
Figure 5. (A) Alizarin red S staining verif ied the for mation of mineralized nodules. (B) Alizarin red S staining verifi ed the formation of mineralized nodules.
adhered to the COL surface. The pores were between 80 μm and 200 μm and were interconnected and irregular. The pore walls were about 1-2 μm thick and the combination of nHAP and COL was in a good condition(Fig. 2A, B).
Porosity, swelling capacity, water absorption capacity, mechanical properties and degradability
Porosity, swelling capacity, water absorption, compressive strength and compressive modulus analysis indicated that the physical properties of the scaff old met the basic requirements for use in tissue engineering30-33)(Tables 1 and 2). One hour after the start of the
experiment, the pH was approximately 7.0. From days 1 to 7, the pH increased gradually, then it stabilized at between 7.3 and 7.4 after 10 days. The low pH in the early stage resulted from residual acetic acid in the COL solution. As this acetic acid evaporated, the pH tended to rise
and achieved the desired physiological value (Fig. 3A).
Samples were removed from the PBS, dried, and freeze-dried, then examined for degradation. At 1 h after the start of the experiment, the degradation rate was close to 0. After a day, the degradation rate showed a gradual increase, but tended to be slow. The degradation rate was 5.06%, 8.93%, 12.10%, 14.65%, 18.47% and 21.18% at 1, 2, 3, 4, 6 and 8 weeks after the start of the experiment(Fig. 3B).
bFGF release from scaff olds
bFGF release from the bFGF/nHAP/COL scaff olds occurred for 17 days. By day 18, the amount of bFGF released into the supernatant by each sample was small (< 1 ng) or undetectable. The total amount of bFGF released by the scaff old was 91.05 ± 3.38%. Between days 3 and 5, the amount of bFGF released from the scaff olds suddenly increased, and then the release continued more gradually after day 5(Fig. 4). Calcifi ed nodules confi rmed the identity of BMSCs
days, microscopy revealed crystalline masses of diff erent sizes. These were calcifi ed nodules that formed early in the diff erentiation process. The proliferation rate of BMSCs obviously decreased over time. Over the course of induction, the number and area of the crystalline masses increased, and dark masses were visible, which were calcifi ed nodules. A small number of nodules were removed and fl oated in the medium during the change of culture medium. After induction, the cells were fi xed with paraformaldehyde and stained with alizarin red S, and red-stained calcifi ed nodules were visible(Fig. 5 and B).
Cell morphology
Cell morphology was observed using an inverted light microscope (CKX41; Olympus). The BMSCs exhibited high survival rates and rapid proliferation. During 6 days of culture, the cells proliferated vigorously, and showed a high density in the early stage and no obvious deformation in passage. Images acquired with an SEM showed cells randomly distributed over the surface and interior of the scaff old. After 3 days of culture with the scaff old, a large number of cells adhered to the scaff old surface and pores. The cell aggregation range was enlarged and the proliferation phenomenon was active (Fig. 6A). Observations with an SEM showed that the single cells displayed polygonal or triangular morphology, and microfi laments were tightly attached to the scaff old (Fig. 6B).
Figure 6. (A) Cell morphology of BMSCs, examined under a light-inverted microscope. The third generation cells exhibited adequate growth and active proliferation, with an increased density observed. (B) SEM images shows that a large number of BMSCs grew on the scaff old, and the morphology was good.
150 100 50 0 A dh es io n ra te ( % ) 1 2 3 Culture time (h)
Figure 7. Adhesion rate of BMSCs cultured with bFGF/nHAP/COL, nHAP/COL scaff old and the control. Data are expressed as the mean ± standard deviation. *P<0.05, vs. the control group; #P<0.05, vs. nHAP/ COL. conttrol group nHAP-COL group bFGF-nHAP-COL group 2.5 2.0 1.5 1.0 0.5 0.0 1 3 5 7 * *# * *# * *#
Culture time (days)
A
45
0
Figure 8. CCk-8 assay determination of the proliferation of BMSCs. bFGF/nHAP/COL, nHAP/COL scaffolds significantly promoted the proliferation of BMSCs compared to the control
conttrol group nHAP-COL group bFGF-nHAP-COL group 15 10 5 0 1 4 7 10 Culture time (days)
* *# * *# *# *
Figure 9. ALP assay of the proliferation of BMSCs cultured with the bFGF/nHAP/COL, nHAP/COL scaffolds and the control group. The bFGF/nHAP/COL scaffold significantly promoted the proliferation of BMSCs after 4 days, compared to nHAP/COL group. Data are expressed as the mean ± standard deviation. *P<0.05, vs. the control group; #P<0.05, vs. nHAP/COL group.
Figure 10. (A) Mandibular defects were prepared by surgical approach. (B) Cells/scaff old or scaff old alone were implanted into the defect area.
control group nHAP/COL group BMSCs/nHAP/COL BMSCs/bFGF-nHAP/COL BMSCs/BMP-2-nHAP/COL BMSCs/BMP-2-bFGF-nHAP/COL normal bone 1000 800 600 400 200 0 C T V al ue ( H U ) a ab abc abc abcde abcde
Figure 12. At 12 weeks after operation, the CT values of no treatment, n HAP/COL alone, BMSCs/n HAP/COL, BMSCs/ bFGF/n HA P/ COL and normal bone were compared. Results are expressed as the means±SD(n=3). aP<0.05, vs. no treatment group; bP<0.05, vs. nHAP/ COL alone group. cP<0.05, vs. BMSCs/nHAP/COL group. dP<0.05, vs. BMSCs/bFGF/nHAP/COL group.
Figure 13. At 12 weeks after operation, the mandibular bone defect area of four groups was observed. (A) The defect area was full of granulation tissue without new bone formation in no treatment group. (B) Granulation tissue mixed with new bone fi lled with the defect area in nHAP/COL alone group. (C) A small amount of granulation tissue and new bone fi lled with the defect area in BMSCs/nHAP/COL group. (D) The defect area has been completely fi lled with new bone and the cortical bone is slightly discontinuous in BMSCs/bFGF/nHAP/COL group.
bFGF promotes BMSC adhesion
The cell adhesion rate was enhanced by increasing the incubation period in three groups. Following culture for 1, 3, and 6 h, the adhesion rate of the scaffold groups was higher than that in the control group (P < 0.05), and the adhesion rate in the bFGF/nHAP/COL group was higher than that in the nHAP/COL group (P < 0.05). Signifi cantly more cells had adhered after 3 and 6 h than after 1 h (P < 0.05). These results demonstrate that bFGF increased BMSC adhesion (Fig. 7).
BMSC proliferation is altered by bFGF
BMSC proliferation in the bFGF/nHAP/COL, nHAP/COL and control groups was compared after 1, 3, 5 and 7 days of culture using a formazan production assay. The absorbance values for the scaffold groups increased, indicating signifi cant cell growth within the scaff olds. The number of cells increased with culture duration in all three groups. BMSC proliferation was observed in all three groups, and there was no signifi cant diff erence between the scaff old groups and the control group after 1 day (P > 0.05). However, after 3 days, the number of cells in the scaffold groups was higher than that in the control group (P < 0.05). Moreover, there was a signifi cant diff erence between the bFGF/nHAP/ COL and nHAP/COL groups (P < 0.05). These results demonstrate that bFGF promoted BMSC proliferation (Fig. 8).
bFGF promotes ALP activity in BMSCs
No signifi cant diff erences in ALP activity were identifi ed between the scaff old groups and the control group during the fi rst 4 days (P > 0.05). However, ALP activity in the scaff old groups was signifi cantly increased compared with that in the control group between days 4 and 10 (P < 0.05). Moreover, ALP activity levels were higher in the bFGF/nHAP/COL group than in the nHAP/COL group (P < 0.05). This suggests that the nHAP-COL scaffold effectively promoted BMSC activity and that bFGF further enhanced this impact. Additionally, the rate of increase in ALP activity in all three groups increased rapidly with increasing culture duration, until the values peaked after 10 days (Fig. 9).
Animal and clinical observations
Mandibular defects were created in twelve rabbits. All of the rabbits exhibited postoperative swelling in the week following the surgery, but no functional consequences of the surgery were observed. No rabbits developed abscesses or had to be sacrificed. Samples were taken 12 weeks after surgery (Fig. 10A, B).
Radiographic analysis
To observe new bone formation and bone defect repair, 3D-CT and X-ray images were acquired and CT values were analyzed 12 weeks postoperatively. This analysis confirmed that more new bone was generated in the BMSC/bFGF/nHAP/COL group than in the other groups. A large amount of granulation tissue without new bone was generated in the control group (Fig. 11).
The CT values revealed that the density of the defect area was signifi cantly higher in the BMSC/bFGF/ nHAP/COL group than in the other groups. (Fig. 12)
Gross observation of specimens
After 12 weeks, the rabbits were sacrifi ced and the mandible was observed. New bone formation in the defect area was most evident in the BMSC/bFGF/nHAP/COL group, compared with the other groups. No new bone formed in the control group; rather, the defect was completely fi lled with granulation tissue (Fig. 13).
Histological analysis
The BMSC/bFGF/nHAP/COL group exhibited the greatest osteogenic effect at 12 weeks, evident as new bone formation in the implanted area, compared with other cell/scaffold or scaffold only groups, and with the control group, in which no new bone was visible. Osteoid matrix formed near the junction of the host bone and the implant, and also formed to a similar extent inside the scaffold and adjacent to the bone wall (Fig. 14).
Discussion
lyophilization34). In tissue engineering, there are many ways to control
the release of factors, for example use of nanoparticles, hydrogels, or porous scaffolds35). Jun et al. effectively achieved controlled release
of growth factors using tissue engineering scaffolds created by lyophilization36). It also demonstrated that lyophilization can produce a
controlled release eff ect. Collagen exhibits good adsorption of protein factors10). Therefore, lyophilized, porous, spongy collagen scaffolds
ensure controlled release of growth factors. During the lyophilization process, the component molecules produce a stronger polymerization force37), thus ensuring maintenance of degradation, and improving the
physical performance of the scaff old to a certain extent.
nHAP has good biocompatibility and has been widely used in artificial clinical materials12). Its addition increases the surface area
of a scaff old, thus increasing the adhesion area. Therefore, an nHAP/ COL scaffold adsorbs bFGF very well and exhibits good controlled release. In the present experiment, bFGF release was sustained over almost 20 days. The traditional form of bone defect regeneration comprises four stages: hematoma formation, hematoma organization, bone callus formation and callus remodeling, and the critical period is the fi rst 3 weeks38). Therefore, the bFGF release duration achieved in
this study essentially meets the physiological requirements for bone defect regeneration. CCK-8 and ALP tests showed that bFGF promoted BMSC proliferation and differentiation. Scaffolds without bFGF also promoted proliferation and differentiation to a lesser extent, thereby demonstrating the non-toxic nature of the scaff old.
Figure 14. Histological analysis of HE staining in four groups at 12 weeks(Light microscope, ×100 magnifi cation): (A) No obvious osteoid matrix, blood vessels and new bone formation in no treatment group. (B) A small amount of new bone was formed and osteoid matrix was visible on normal bone margin in nHAP/COL alone group. (C) More new bone was formed, more osteoid matrix was distributed between new bone and normal bone, and new blood vessels was visible in BMSCs/nHAP/COL group. (D) More new blood vessels was found, new bone and normal bone have formed a certain combination in BMSCs/bFGF/nHAP/COL group. M, mandible; O, osteoid matrix; NV, new blood vessel; NB, new bone.
The biocompatibility of the bFGF/nHAP/COL scaffold has been well documented in vitro, and we further studied this in vivo. When a bone defect reaches a certain range, no new bone forms through the normal physiological healing process; this is considered a critical bone defect19). The practical signifi cance of this to bone tissue engineering
is that when a critical bone defect occurs, new bone can be induced by introduction of a biological scaffold seeded with cells and loaded with growth factors to regenerate the bone defect39). During bone
formation, the scaff old is degraded continuously, and the product is not biologically toxic. Allogeneic BMSCs, which are not immunogenic, are ideal seed cells17).
our results confi rmed an obvious osteogenic eff ect. Taken together, our results indicate that the bFGF/nHAP/COL scaff old has good physical properties and biocompatibility in vitro, and promotes osteogenesis in
vivo.
Ethical approval and consent to participate
In the experiment, 12 healthy New Zealand rabbits (both male and female, weighing about 2.5kg) were obtained from the experimental animal center (Shenyang, China) of China Medical University. Animal license No. scxk-ln2011-0009, animal use agreement No. cmu62043006. All experiments and surgical procedures were approved by the China Medical University Committee on animal care and use, and were followed by the National Institutes of health's laboratory animal care and use guidelines. All eff orts were made to minimize the number of animals used and their suff ering.
Acknowledgements
The current study was supported by the science and technology plan project of Liaoning province [grant number 00002012225082]. YC reviewed the literature and wrote the manuscript. XKW was a contributor in reviewing the literature. XXT, LZ and RZ, TZ, YD revised the manuscript. All authors read and approved the final manuscript.
Confl ict of interests The authors have no COI existed.
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