The healing of bone defects with large gaps is the problem of many patients. Using biological scaffolds or nonbiodegradable synthetic compounds with unique properties have been able to repair small defects.1–4 Human umbilical cord blood (UCB) is a rich source of hematopoietic stem cells for clinical application and may be one of the wide sources of stem cells with naive immune status.5,6 Cord lining mesenchymal stem cells (MSCs) express CD23, CD14, and low amounts of CD34 and CD35; they are not able to express endothelial marker CD31 and have greater expansion in vitro than Wharton’s jelly-derived MSCs.7 CD14 inhibits T cells. Wharton’s jelly-derived MSCs do not express CD14 or CD23. Despite those descriptions, the cell markers of umbilical cord-derived MSCs still remain under consideration.8,9 One of the key factors of tissue engineering is to prepare a three-dimensional (3D) scaffold with suitable properties and also, high porosity, interconnected pores, and others. In natural tissues, cells are surrounded by extracellular matrix, which has a physical structural features ranging from nanometer scale to micrometer. To mimic the natural extracellular matrix, many studies have been done to fabricate nanostructured scaffolds.10–12 Polyhydoxyalkanoates (PHAs) are polyesters produced by microorganisms under unbalanced growth conditions. PHAs are generally biodegradable, with good biocompatibility, making them attractive as tissue engineering biomaterials. Only several PHAs, including poly 3-hydroxybutyrate (PHB), copolymers of 3-hydroxybutyrate and 3-hydroxyvalerate (PHBV), poly 4-hydroxybutyrate (P4HB), copolymers of 3-hydroxybutyrate and 3-hydroxyhexanoate (PHBHHx), and poly 3-hydroxyoctanoate (PHO), are available in sufficient quantity for application research.13–29 Hydroxyapatite [HAp, Ca10(PO4)6(OH)2] is a type of calcium phosphates, which has extensive applications in the healing of bone and tooth, because of biocompatibility and similar composition to natural bone.30,31 In the current study, unrestricted somatic stem cells (USSCs) were used as a regenerative cell for the growth and repairing of bone defect. In fact, USSCs are one of the rare cell populations in UCB, which is considered as pluripotent. Moreover, USSCs are highly potential for proliferation and differentiation. Therefore, USSCs are valuable sources for cell therapy.32,33 The purpose of this study was to investigate the bone-healing effects of scaffold containing PHBV and HAp nanoparticles combined with USSCs in calvarial bone of rats.
Design of Nanofibrous Scaffolds
The HAp, Ca10(PO4)6(OH)2, nanopowders (<200 nm) were purchased from Sigma-Aldrich Co (St. Louis, MO). A PHBV containing 5 mol% of 3-hydroxyvalerate with 680,000 molecular weight was purchased from Sigma Chemical Co. To prepare PHBV solution, 2,2,2-trifluoroethanol (TFE) was also purchased from Sigma-Aldrich Chemicals and was used as received without further purification. Electrospinning apparatus used in this study was prepared from Nano Azma Company (Tehran, Iran). Poly (3-hydroxybutyrate-co-3-hydroxyvalerate) (2% W) was dissolved at determined concentration in TFE. The nanoparticle powders sterilized by autoclave were added (5%, 10%, and 20wt%) directly to the above stirring solution (with ultrasound, 60 W) to the resulting solution to obtain a homogeneous liquid solution of nanoparticles HAp/PHBV. The PHBV solution was contained in a glass syringe controlled by syringe pump. A positive high voltage source through a wire was applied at the tip of a syringe needle. In this situation, a strong electric field (20 kV) is generated between PHBV solution and a collector. When the electric field reached a critical value with increasing voltage, mutual charge repulsion overcame the surface tension of the polymer solution and an electrically charged jet was ejected from the tip of a conical shape as the Taylor cone. Ultrafine fibers are formed by narrowing the ejected jet fluid as it undergoes increasing surface charge density because of evaporation of the solvent. An electrospun PHBV nanofibrous mat was carefully detached from the collector and dried in vacuum for 2 days at room temperature to remove solvent molecules completely. The nanofibers were designed with determined parameters (syringe size, 17 mm; collector speed, 1000 rpm; injected speed, 2 ml/min; syringe tip distance to collector,: 75 mm; voltage, 20 kV; temperature, 30°C; time, 7 h).
Characterization of Nanofibrous Scaffold
The surface characteristics of various scaffolds were studied by scanning electron microscopy (SEM; Cambridge Stereo-scan, model S-360; Cambridge Instruments, Wetzlar, Germany) to analyze the changes in the surface morphology. The scaffolds were first coated with a gold layer (Joel fine coat, Joel LTD., Tokyo, Japan, ion sputter for 2 h) to provide surface conduction before their scanning. The X-ray diffractometer (XRD, Thermo ARL XTRA) was used to determine the phase structure of the PHBV and PHBV/HAp nanofibrous scaffolds. The XRD patterns were obtained in a 2θ angle ranging from 10° to 60° at a scanning speed of 2°/min.
Culture and isolation protocol of the USSC from fresh UCB was previously described by Kögler et al.33 After consent from mothers, their umbilical cords were obtained from the cord vein. Only 40% of the cord blood samples contained USSCs. The mean age of donors was 28 years. After collecting the samples, the red blood cells were lysed using ammonium chloride (NH4Cl), and the isolation procedure was continued by Ficoll (Amersham, United Kingdom). Then the samples were rinsed twice with sterile phosphate-buffered saline (PBS, pH:7.4). After centrifuging, the resultant cells were placed in Dulbecco’s modified Eagle’s medium (DMEM) low glucose, which had been enriched with 100 nmol dexamethasone, 10% fetal bovine serum (FBS), penicillin, and streptomycin. The first medium exchange process was done for 24 h, then every 4 days. When 80% of the flask surface area was covered by the cells, the cells were passed by using 0.25% trypsin and ethylenediaminetetraacetic acid (EDTA). The USSCs were regularly expanded on the culture medium, and the 37°C temperature and 5% of CO2 were required for the growth. The adherent cells were detached with 0.25% trypsin/EDTA, centrifuged for 5 min at 1200g and resuspended in PBS. Cell proliferation and viability in vitro were analyzed with the tetrazolium salt 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT). The yellow dye MTT is reduced to a blue formazan product by respiratory enzymes that are active only in viable cells, making the amount of color change indicative of cell proliferation. Five thousand cells of human USSCs were seeded on nanofibrous PHBV scaffolds. For analysis, 20 μl of MTT (sigma) substrate (of a 2.5 mg/ml stock solution in PBS) was added to each well, and the plates were returned to standard tissue incubator conditions for an additional 4 h. Medium was then removed, the cells were solubilized in 100 μl of dimethyl sulfoxide, and colorimetric analysis was performed (wavelength, 570 nm, RAYTO microplate reader, Shenzhen, China). For SEM study, the scaffolds with cells were washed by PBS and then fixed by glutaraldehyde (2.5%) at 4°C for 2 h. The samples were dehydrated by alcohols and then kept with tetraoxide osmium vapors at 4°C for 2 h. The samples were kept in desiccators, coated with gold, and investigated by a SEM (Cambridge Stereo-scan, S-360).
Alkaline Phosphatase Activity
The samples were washed with PBS solution and transferred to 1.5 ml microtubes containing 1 ml ultra-pure water. Cell constructs were cryopreserved at −80°C, for further analysis. Before alkaline phosphatase (ALP) quantification, the samples were thawed and were sonicated for 15 min. The ALP activity was measured by the specific conversion of p-nitrophenylphosphate (pNpp; Sigma) into p-nitrophenol (pNp). The enzymatic reaction was set up by mixing 100 ml of the sample with 300 ml of substrate buffer containing 1 M diethanolamine HCl, pH 9.8 and 2 mg/ml pNp. The solution was further incubated at 37°C for 1 h, and the reaction was stopped by adding a solution containing 2 M NaOH and 0.2 mM EDTA. The optical density (OD) was determined at 405 nm.
A total of 30 male Wistar white rats aged ~4–8 weeks at the beginning of the experiment and weighing 180–220 g were divided into three groups, each group containing 10 animals. The protocol for the experiment was approved by the Institutional Animal Care and Use Committee of Beheshti University (Iran). Animals were handled according to the guidelines established for animal care at the center. The calvarial defect model is convenient for examining bone regeneration because of its effective accessibility and lack of fixation requirements. Critical size defect in experimental models is essential bone reconstruction in in vivo experiments34,35; also, the research and development on critical size defect attracted more attention in recent years.36 Cavity of approximately 5 mm wide, 5 mm long, and a depth of 8 mm was created by trephine bur (Tokyo, Japan) in calvarial bone of rats. After preparation of all groups, the cells were harvested from tissue culture petri dish, were centrifuged (200g), and were mixed by rotator shaker. Last, this functional combination was pumped in to the bone defect. Finally, 2 × 106 USSC per defect were used. The defects were filled with the designated multilayer scaffolds loaded with or without stem cells and skin sutured using 5–0 nylon at 0.75 cm intervals. Among the 30 bone defects, 10 defects were filled with PHBV/nano-HAp (20%) scaffold without cell (group A), 10 defects were filled with PHBV/nano-HAp (20%) scaffold with cell (group B), and 10 defects were treated as control group (group C), and then each animal was housed in its own cage to avoid damage to the wound. Postsurgery care included analgesic and antibiotic (cyclosporine) injections. Checking of any postsurgery pain, distress, or complications was done 24 h after surgery and daily afterward. Figure 1 shows process of bone reconstruction with USSCs and scaffold.
Computed Tomography Analysis
A computed tomography (CT) scanner (3 T, Siemens Sensation 16 Slice CT, Erlangen, Germany) was used to determine bone growth occurring in the engineered constructs. Specimens were scanned with a spatial resolution: 30 lp/cm and reconstruction time per image was 0.1 s. Samples were also analyzed using syngo software and therefore will identify areas of bone density.
For observation of the tissue structures and thickness of the new bone, histological examinations were carried out. Samples were fixed with formalin 10% for 2 weeks. Subsequently, the samples were processed for histological examination according to standard procedures. The samples were embedded in paraffin, sectioned at 4 μm thickness and subjected to hematoxylin and eosin (H&E) staining. All the stained sections were observed by optical microscope (Nikon, Tokyo, Japan). After 4 weeks, the reconstructed area was analyzed by histomorphometric measurements of the width of the repaired area, and then the correlation coefficient (r = 1,0; p < 0.05) and detected rebuilding rate were calculated. The reconstructed area <20% (faulty healing), reconstructed area between 20% and 60% (partially healing), and >60% (significant healing) were defined in this analysis. Experimental results were expressed as means ± S.D. All data were analyzed by one-way analysis of variance (ANOVA) with Duncan’s multiple range tests (Duncan multiple range test, p < 0.05 and 0.01).
Results and Discussion
Characterization of Nanofibrous Scaffold
Nanofibrous scaffold designed by electrospinning method (Figure 2). In composite materials, the surface and inner structure and distribution of the components have a decisive impact on the materials properties. For tissue engineering application, distribution of the HAp in-and-outside the fibers will further influence cytocompatibility of the scaffolds, such as cell adhesion and proliferation. Therefore, morphology and structure of the pure PHBV and PHBV/nano-HAp electrospun scaffolds were observed under SEM, as shown in Figure 3. Both the control PHBV and PHBV/nano-HAp electrospun scaffolds showed a randomly interconnected and highly porous structure, which was composed of continuous bead-free nonwoven nanofibers (Figure 3). The fiber diameter increased from 100 ± 117 (for the PHBV) to 124 ± 110 nm and further to 300 nm after addition of 5% HAp and higher amount of HAp, respectively (Table 1). Compared with a smooth morphology of the control PHBV nanofibers (Figure 3A), rough surfaces were observed for all the composite scaffolds (Figure 3B–D). A few tiny particles were found on the fiber surfaces when 5% nano-HAp was added (Figure 3B). Addition of 10% nano-HAp resulted in a structure with some nanoparticle protuberances on the fiber surfaces (Figure 3C), when the amount was further increased to 20% nano-HAp, the surface of the fibers was even entirely covered by a layer of nanoparticles (Figure 3D). In this regard, the electrospun composite nanofibers of PHBV and HAp could better mimic the micro-/nano-structure of the natural bone.37
Table 1 shows physical properties of designed PHBV nanofibrous scaffolds. The pore size of PHBV nanofibrous scaffolds was measured approximately 0.40 μm. However, the pore size of all the scaffolds remained more or less constant with an average value of 4500 nm regardless of the HAp addition. This is quite common for the electrospun fibrous scaffolds, in which the pores are formed by the injected fibers and thereby can be only varied within a small range. This is consistent with a previous study, in which the pore size was not significantly varied, although the diameter of nanofibers was decreased along with the increase of voltage.38
To study the crystalline structure of the HAp in the nanocomposite, XRD characterization was performed (Figure 4). Appearance of the broad peaks in the 2θ = 20–60° region was attributed to the amorphous nature of the PHBV polymer (Figure 4A). After incorporation of HAp, the patterns of the composite scaffolds (here taking the PHBV/20% HAp nanofibers as a typical example) exhibited several peaks at 2θ = 25–29°; 30–34°; and 46.7° which correspond to (002), (102), (210); (211), (112), (300), (202); and (222) typical spectrum of HAp (Figure 4B), respectively. Spectra can conclude that the crystalline structure of the HAp had been preserved in the nanofibrous composite scaffolds.
Table 2 shows the MTT assay for tissue culture polystyrene (TCPS; control group) and the nanofibrous scaffolds. The results showed a high viability for the nanofibrous samples. Also, the nanofibrous samples caused more USSCs to proliferate. Figure 5 shows images of the cell culture on the nanofibers and the control sample. Figure 5A is related to the control sample and Figure 5B is related to the PHBV nanofiber. Cellular images showed good growth in the vicinity of nanofibers.
Cell Differentiation and ALP Activity
The ALP activity of human USSCs cultured onto the scaffolds did not follow the typical trend of this marker of osteogenic differentiation, as it was demonstrated after 21 days. After this time point, a significant increase in ALP activity was observed (Figure 6). Usually, ALP reaches a peak at an earlier time point.
After 4 weeks, before the CT-scan, the rats were anesthetized with xylene and ketamine and then were placed inside the inhibitor box, and rebuild area was evaluated by CT-SCAN (3 T). The figures show reconstructing coordination with the scaffolds. The CT results suggested that after 4 weeks of implantation, cell constructs promoted bone regeneration of the calvarial defect (Figure 7). The CT images also supported the enhanced bone ingrowth in scaffolds cultured with USSCs. Some of the images clearly show an almost complete healing of the defect (Figure 7C and F). Scaffolds with cells were able to induce some bone regeneration/ingrowth. New bone formation could be because of invading reparative cells from the dura or from adjacent host tissues. The selected implantation time seemed to be adequate for assessing the complete bone healing at the site defect, as shown in Figure 7C. Table 3 shows regeneration of calvarial bone of rats that were obtained by CT analysis.
Typical H&E-stained histological sections at 4 weeks after implantation are shown in Figure 8. After 4 weeks, in control sample, fibrous tissue extends from the defect margin with a few small areas of new bone formation (≤10%) visible immediately adjacent to the host bone, indicating unsuccessful efforts at regeneration, whereas histomorphometric results showed that >55% reconstruction has occurred in calvaria for the scaffold with cells after 28 days. The new bone formation was both continuous and discontinuous with the calvarial bone. The new bone was thin, and the tissue structure was incomplete for the scaffold. Bone formation increased for the scaffold/USSC group. The thickness and structure of the new bone were better in the scaffold/USSC group than the scaffold group. In the scaffold group without cells, the continuity of the bone regeneration was incomplete. Ossification rate for the samples with USSC is more than the other groups, and bone quality and relevance of new bone tissue and surrounding bone tissue were established for the scaffold/USSC group. None of the samples showed any marked inflammatory reactions caused by the bonding agent or other materials. The grafted scaffold was absorbed and replaced by new bone, which reached the level of the periosteal surface of the defect and surrounded the grafted scaffold (Sc). Enclosed areas in panels (D), (E), and (F) of Figure 8 are shown as magnified images in panels (G), (H), and (I), respectively. The regenerated bone in panel (I) shows a histological profile of compact bone featuring cement lines (black arrows) strongly stained with hematoxylin.
In this study, we evaluated the ability of PHBV/nano-HAp scaffolds with and without USSCs to promote bone healing in critical size rat calvarial defects. Previous studies revealed that natural based polymers, such as chitosan,29 are highly potential for bone tissue engineering applications. Nanosized HAp and its derivatives may have other special properties because of its small size and large specific surface area.39 Webster et al.40 demonstrated a significant increase in protein absorption and osteoblast adhesion on the nanosized ceramic. The advantage of paste or injectable calcium phosphates gel is that it is freely moldable and adaptable to bone defects. The combination of high biocompatibility, easy-to-shape characteristic, and the capacity to self-set under ambient conditions makes it an asset in repairing hard tissue defects.41,42 Chen et al.43 showed an increase in osteogenic markers production on chitosan/nano-HAp/collagen scaffolds. Their results represent the used potential of scaffolds in bone tissue engineering.
Zhang et al.44 developed an injectable nano-HAp/chitosan scaffold for repairing rabbit femoral condyle bone defects with critical size bone defects (6 mm in diameter and 10 mm in length). Twelve weeks after surgery, complete healing of the segmental bone defect was observed in the nano-HAp/chitosan group, whereas the defect was still visible in the chitosan group, although the depth of the defect had diminished. These observations suggested that the PHBV scaffolds were potential candidate materials for regeneration of bone loss. The combination of scaffold with stem cells is considered as a useful method for bone regeneration. Bone marrow-MSCs (BM-MSCs) are the main cell source in bone tissue engineering applications because of their capabilities for self-renewal and osteogenic differentiation potential both in vitro and in vivo.45–47 Histological analysis complemented the micro-CT findings of bone formation within defects. Cross-sectional analysis of the defects demonstrated a greater amount of bone growth centrally in those defects containing MSC rather than MSC-free defects. In 4 weeks, the amount of bone healing or regeneration obtained was approximately 20% for gel alone and approximately 30% for scaffold/BM-MSCs.48 Diao et al.49 developed a 3D porous biomimetic scaffold, which is composed of HA, collagen, and poly-L-lactide acid (PLLA) with a cube shape (5 × 5 × 5 mm3), with UCB-MSC. Analyses revealed that in cell-free implant no bone-like tissue was formed during the entire healing period. Collectively, these results represent successful in vivo osteogenesis by UCB-MSCs. In another study, UCB-MSCs were mixed with an HAp/tricalcium phosphate (TCP) composite and were implanted into the subcutaneous region of immunodeficient mice for 8 weeks, and ectopic bone formation was observed on the surface of the HAp/TCP composite material.50,51 The coverage of the defect with bone was not an unexpected event, especially with the addition of cells. The cellular group increased the amount of bone formation at the periphery of the defect, whereas the MSCs provided a supply of cells and elaborated the factors that generated bone closer to the center. In our study, no statistically significant difference was found between the control group and the scaffold alone group at the 4 week time points. This is in stark contrast to the MSC/scaffold group, which had significant bone healing at 4 weeks. The analysis also quantitatively revealed that the amount of bone generated at the end of 4 weeks was approximately four times more in the group that received MSC when compared with all other groups (approximately 20% for scaffold alone [faulty healing] and approximately 60% for scaffold/USSCs [partial healing]). Our obtained results with scaffold/USSCs were better than Stephan et al.48 study with scaffold/BM-MSCs, On the other hand, the acquisition of BM-MSCs from bone marrow is an invasive procedure and can create complications, such as infection, bleeding, and chronic pain to donors. Moreover, the number and differentiating potential of BM-MSCs in bone marrow and their ability to differentiate decrease with age, thus limiting the wide application of bone marrow stromal cells (BMSCs) in tissue engineering and cell-based therapeutics.52–54 Umbilical cord stem cells have been found to be less immunogenic than adult marrow and blood cells,55 suggesting that allogenic UCB-MSCs obtained from more than one single cord might be used for bone tissue-engineering in patients.
A major limitation to regeneration of bone is the relative lack of a vascular supply. A diffusion barrier exists beyond which cells are unable to obtain nutrients via plasmatic imbibition. Thus, much of the bone that is produced has a limited diameter directly related to the size at which the cells cannot support themselves through diffusion. Histological analysis complemented the micro-CT findings of bone formation within defects. Cross-sectional analysis of the defects demonstrated a greater amount of bone growth centrally in those defects containing MSC compared with MSC-free defects. Another limitation of this study is the relatively low frequency of MSCs in full-term UCB. During fetal development, the process of hematopoiesis, which is initiated within the yolk sac before moving through the circulation to other anatomical sites within the developing embryo, there is an inverse correlation between gestation time and the frequency of both hematopoietic stem cells and MSCs within UCB.56–59 Therefore, further investigation for rapid and efficient methods for isolating and expanding MSCs from full-term UCB are needed. In addition, this study demonstrates that BM-MSC can be replaced with USSCs for bone regeneration, thus also indicating that UCB-MSCs with PHBV/nano-HAp scaffold as an osteoconductive and osteoinductive agent might possibly serve as a suitable combination for bone regeneration.
In the current study, the bone-healing effects of PHBV/nano-HAp nanofibrous scaffold with USSCs grafted in rat calvarial model were investigated. In addition, bone regeneration was achieved using the injectable scaffold especially with stem cells in rat calvarial bone. This study evidenced very positive results that highlight the possibility of scaffolds especially import effect of USSCs to be used as effective design for healing non–load-bearing bone defects.
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