In the preliminary study, we have found an excellent osteogenic property of nanohydroxyapatite/chitosan/poly(lactide-co-glycolide) (nHA/CS/PLGA) scaffolds seeded with human umbilical cord mesenchymal stem cells (hUCMSCs)
Scaffold materials for bone tissue engineering are usually divided into inorganic materials, natural biomaterials, and synthetic polymer materials [
In recent years, it has been reported that hUCMSCs have similar characteristics with bone marrow-derived mesenchymal stem cells (BMSCs). It might be used as a selection of seed cells for bone tissue engineering [
In our preliminary study, when hUCMSCs were seeded onto nHA/CS/PLGA scaffolds the results showed higher osteoinduction activity compared with hUCMSCs seeded onto nHA/PLGA, CS/PLGA, or PLGA scaffolds
Nevertheless, the capacity of repairing bone defects is a key factor to assess the osteogenic ability in bone tissue engineering. Calvarial defect models of nude mice are used commonly in bone tissue engineering [
nHA/CS/PLGA scaffolds were prepared as described in the preliminary study [
hUCMSCs were obtained as described in the preliminary study [
Totally 108 nude mice (NIH strain, outbred, 8-week-old males) were preanesthetized with pentobarbital sodium (30 mg/kg) via intraperitoneal injection. The subcutaneous tissue, musculature, and periosteum were dissected to expose the calvarium. Full-thickness defects of 6 mm in diameter were created in the central areas of cranial bones using a saline-cooled trephine drill (Appledental, Hong Kong, China). After being maintained in an osteogenic medium for 2 weeks, the scaffolds seeded with hUCMSCs were implanted into the calvarial defects. Six groups were randomly divided according to the defects implanted with different contents: (1) PLGA scaffolds + hUCMSCs; (2) nHA/PLGA scaffolds + hUCMSCs; (3) CS/PLGA scaffolds + hUCMSCs; (4) nHA/CS/PLGA scaffolds + hUCMSCs; (5) nHA/CS/PLGA scaffolds without seeding; (6) the control group (no scaffolds) (
The images in calvarial defects of nude mice. (a) The calvarial defects of 6 mm in diameter. (b) The scaffolds were implanted into the calvarial defects.
In order to show the mineralization areas of new bones, sequential fluorescent labeling was performed by giving intracutaneous injections with xylenol orange (90 mg/kg, Xi’an Chemical Reagent Factory, China), fluorescein sodium (3 mg/kg, Xi’an Chemical Reagent Factory, China), and tetracycline (50 mg/kg, Xi’an Chemical Reagent Factory, China) on the back of nude mice at 1, 2, and 3 weeks after operation. At 4 weeks after operation, 12 nude mice from each group were euthanized by using the overdose of sodium pentobarbital, and the calvarial specimens with 2 mm of contiguous bones were then removed.
The nondecalcified calvarial specimens from the 6 nude mice in each group (
After decalcified in 14% ethylenediaminetetraacetic acid (EDTA) for 8 weeks, the 6 calvarial specimens from each group (
At 8 weeks, the remaining nude mice were euthanized, and the samples of six nude mice in each group were obtained by the above-mentioned method. Briefly, the nondecalcified samples were embedded by methyl methacrylate and cut into the sections of 80
All data were analyzed using statistical software SPSS17.0 and expressed as means ± standard deviation. The significance tests were performed by one-way analysis of variance (ANOVA), followed by Bonferroni
At 4 weeks, all nude mice acted and ate normally after operation. The operation areas showed mild swellings. The incisions were not infected and healed well. Rejections were not observed in all groups. The images of sequential fluorescent labeling exhibited fluorescent marks around the defects, except in the control group. Red, yellow, and green fluorescence bands were irregularly scattered as strips, rods, and bulks. No obvious boundaries were detected between those fluorescence bands. In the group of nHA/CS/PLGA scaffolds + hUCMSCs, the fluorescent labeling was visible at the edges and centers of the defects and the fluorescence-labeled areas showed more extensive signals compared with the other groups (
The percentages of fluorescence-labeled areas at 4 weeks after operation (
Groups | nHA/CS/PLGA scaffolds + hUCMSCs | nHA/PLGA scaffolds + hUCMSCs | CS/PLGA scaffolds + hUCMSCs | PLGA scaffolds + hUCMSCs | nHA/CS/PLGA scaffolds without seeding | The control group |
---|---|---|---|---|---|---|
The percentages of fluorescence-labeled areas (%) | 46.33 ± 9.07 |
31.66 ± 18.72 | 25.33 ± 8.73 | 21.33 ± 7.02 | 11.00 ± 4.58 | 1.67 ± 3.78 |
The percentages (%) of fluorescence-labeled areas in nHA/CS/PLGA scaffolds + hUCMSCs group were higher than those of other groups,
The images of sequential fluorescent labeling at 4 weeks. (a) nHA/CS/PLGA scaffolds + hUCMSCs; (b) nHA/PLGA scaffolds + hUCMSCs; (c) CS/PLGA scaffolds + hUCMSCs; (d) PLGA scaffolds + hUCMSCs; (e) nHA/CS/PLGA scaffolds without seeding; (f) the control group (no scaffolds). The fluorescent labeling was visible around and inside the defects and the fluorescence-labeled areas were more extensive compared with the other groups (
H&E-stained sections of calvarial defects at 4 weeks are shown in Figure
The percentages of the osteoid tissues and bone islands at 4 weeks (
Groups | nHA/CS/PLGA scaffolds + hUCMSCs | nHA/PLGA scaffolds + hUCMSCs | CS/PLGA scaffolds + hUCMSCs | PLGA scaffolds + hUCMSCs | nHA/CS/PLGA scaffolds without seeding | The control group |
---|---|---|---|---|---|---|
The percentages of the osteoid tissues and bone islands (%) | 51.50 ± 4.69 |
32.63 ± 3.79 | 28.73 ± 3.18 | 27.09 ± 3.23 | 21.32 ± 2.88 | 0 |
The percentages (%) of the osteoid tissues and bone islands in nHA/CS/PLGA scaffolds + hUCMSCs group were higher than those of other groups,
HE-stained sections of calvarial defects at 4 weeks. (A) nHA/CS/PLGA scaffolds + hUCMSCs; (B) nHA/PLGA scaffolds + hUCMSCs; (C) CS/PLGA scaffolds + hUCMSCs; (D) PLGA scaffolds + hUCMSCs; (E) nHA/CS/PLGA scaffolds without seeding; (F) the control group (no scaffolds). (G), (H), (I), (J), and (K) were the enlarged photographs of the rectangular frames in (A), (B), (C), (D), and (E), respectively. The bone islands were scattered around and inside the scaffolds (A). The bone islands could be occasionally observed around the defects. The osteoid tissues could be found around and inside the defects (B, C, and D). However, in the group of nHA/CS/PLGA scaffolds without seeding, osteoid tissues were only found around the defects, and bone islands were not detected (E). No osteoid tissues or bone islands were observed in the control group (F). BI: bone islands, OT: osteoid tissues, and S: scaffolds. Original magnification (A–F): 50x. Original magnification (G–K): 300x.
At 8 weeks, in the group of nHA/CS/PLGA scaffolds + hUCMSCs, the defects were filled with new bones. No obvious boundaries were found between the new bones and host bones. In the other groups except for the control group, the defects were partially healed. Similarly, no obvious boundaries were found between the new bones and host bones (Figure
Macroscopic observation of the defects at 8 weeks after operation. (a) nHA/CS/PLGA scaffolds + hUCMSCs; (b) nHA/PLGA scaffolds + hUCMSCs; (c) CS/PLGA scaffolds + hUCMSCs; (d) PLGA scaffolds + hUCMSCs; (e) nHA/CS/PLGA scaffolds without seeding; (f) the control group (no scaffolds). The defects were filled with new bones (a). No obvious boundaries were found between the new bones and host bones (a–e). The defects were partially healed in the control group (f).
Van Gieson staining in the calvarial defects at 8 weeks. (a) nHA/CS/PLGA scaffolds + hUCMSCs; (b) nHA/PLGA scaffolds + hUCMSCs; (c) CS/PLGA scaffolds + hUCMSCs; (d) PLGA scaffolds + hUCMSCs; (e) nHA/CS/PLGA scaffolds without seeding; (f) the control group (no scaffolds). Most of bone defects were replaced by new bones and scaffolds were found degraded mostly. There was no difference between new bones and host bones. There were lots of mature and cord-like lamellar bones bridged with host bones (a). The remaining scaffolds could still be observed. New block-style and island-style bones formed inside the defects (b, c, d, and e). New bones were visible around the defects but invisible inside the defects (e). There were no new bones found in the control group (f). NB: new bone; S: scaffold. Original magnification: 300x.
New bones were the most in the group of nHA/CS/PLGA + hUCMSCs (
The percentages of new bones at 8 weeks (
Groups | nHA/CS/PLGA scaffolds + hUCMSCs | nHA/PLGA scaffolds + hUCMSCs | CS/PLGA scaffolds + hUCMSCs | PLGA scaffolds + hUCMSCs | nHA/CS/PLGA scaffolds without seeding | The control group |
---|---|---|---|---|---|---|
The percentages of new bones (%) | 74.30 ± 6.52 |
61.66 ± 2.73 | 55.29 ± 8.76 | 49.46 ± 6.21 | 32.23 ± 4.67 | 0 |
The area percentages (%) of new bones in nHA/CS/PLGA scaffolds + hUCMSCs group were higher than those of the other groups,
Nude mice lack the thymus, resulting in the obstacle of generating T cells. Therefore, they are widely used in the experiments on immunology, oncology, toxicology, and other subjects of life science. Moreover, they have poor blood supply and bone regeneration. Immunosuppression of nude mice also causes their lack of key signals in the process of bone repairing, leading to retarded bone regeneration [
Sequential fluorescent labeling has been widely used in the researches on bone metabolism. The beginning of calcification and the formation of new bones are confirmed qualitatively by the method [
Because the nondecalcified samples at 8 weeks were too hard to be made into fluorescent-labeled sections due to high degree of calcification, therefore the samples only at 4 weeks were made into the fluorescence-labeled sections. In the study, our data showed obvious difference by fluorescent markers between the group of nHA/CS/PLGA scaffolds + hUCMSCs and the other groups, suggesting that the formation of new bones in the group of nHA/CS/PLGA scaffolds + hUCMSCs was faster, compared with the other groups.
The bone samples for HE staining must be demineralized. The nucleus of cells at 8 weeks was stained as blue rather than red, because the samples at 8 weeks needed to be decalcified for a long time. So the sections of samples at 8 weeks were stained by Van Gieson instead of H&E.
It was reported that the scaffolds without seeding repaired bone defects mainly through bone conduction [
Degradability of scaffold materials is crucial for materials in bone tissue engineering. Growth factors secreted by seeded cells induce the aggregation of the osteoclasts via signal conduction. In addition, these growth factors lead to the degradation of materials [
As a kind of ideal inorganic bioactive material, the particle sizes of nHA are small and its surface areas are large. Unique surface properties of nHA, such as chemical properties, surface topography, and surface energy, mediate the bioactivity of specific proteins, such as fibronectin, vitronectin, and laminin. Consequently, cell behaviors are regulated, and the tissue regenerations are further promoted [
It was reported that it was difficult to repair critical calvarial defects in a short time [
Our findings revealed that nHA and CS enhanced the bone regeneration of nHA/CS/PLGA scaffolds seeded with hUCMSCs in the calvarial defects of the nude mice at early stage. nHA/CS/PLGA scaffolds seeded with hUCMSCs could have wide applications in bone tissue engineering.
The authors declare that there is no conflict of interests regarding the publication of this paper.
Fei Wang and Xiao-Xia Su contributed equally to this study.
This study was supported by Natural Science Foundation research project of Shaanxi Province (2014JM4146) and the Fundamental Research Funds for the Central Universities (the Fundamental Research Funds for the Special Research Projects of Xi’an Jiao Tong University: 2015.01–2017.12).