Local transplantation of mesenchymal stem cells (MSCs) is one of the effective therapies for bone regeneration in orthopedics and dentistry [1
]. MSCs have pleiotropic actions including recruitment of other MSCs and progenitor cells, angiogenesis, immunoregulatory effects, anti-apoptotic effects, and promotion of host-cell differentiation into tissue-forming cells [3
]. Transplantation of naive MSC is therapeutically purposed to modulate the regenerative microenvironment [4
], whereas implantation of cultured osteoblasts derived from MSCs is also explored to supply bone-forming cells with osteoinductive properties [7
The effectiveness of cell transplantation on tissue regeneration still has room for improvement [11
]. One of the critical issues is loss of transplanted cell viability after local transplantation [1
]. Regardless of the types of donor cells or recipient tissue, the number of transplanted cells is reduced by more than 50% in 3 days after transplantation, and the cells barely maintain trackability for 2–3 weeks [1
]. The reduced number of transplanted cells compromises the effectiveness of tissue regeneration [16
]. In addition, loss of viability of transplanted cells fails to track the fate of the transplanted cells in local tissue. Locally transplanted osteoblast-like cells in a femoral bone defect were found to remain at the transplantation site without migrating to other organs, but showed no evidence to participate in bone regeneration [18
]. Improvement of viability of transplanted cells would lead to not only enhancing the effectiveness of tissue regeneration but also clarifying the cell fate after local transplantation.
Physical tissue damage in association with transplantation procedures activates innate immune cells such as neutrophils and evokes acute inflammation by producing various proinflammatory mediators, cytokines, and reactive oxygen species (ROS) [19
]. Excessive ROS generation causes oxidative stress on transplanted cells by disturbing the intracellular redox balance, eventually inducing apoptosis [5
]. Oxidative stress resulting from noninfectious acute inflammation can cause the elimination of transplanted stem cells [6
]. In addition, oxidative stress hinders the osteogenic differentiation of MSC and osteoblast progenitors [21
]. The lineage commitment of MSCs is regulated toward adipocytes rather than osteoblasts under oxidative stress [22
]. Addition of exogenous hydrogen peroxide (H2
) suppressed the osteogenic differentiation of cultured human bone marrow-derived MSCs (BMSCs) [23
] and murine osteoblast progenitors [24
]. Regulation of cellular redox balance is one of the key factors to prevent loss of viability and dysfunctions of transplanted cells after local transplantation.
-cysteine (NAC) is an amino acid-derivative with intra- and extracellular antioxidant capabilities [25
]. The functional moiety, the sulfhydryl group, of NAC can directly scavenge extracellular ROS or certain cytotoxic substances. In addition, NAC undergoes cellular uptake through solute carrier transporters [26
] and deacetylation within the cytoplasm into a precursor, L
-cysteine, of a major intracellular antioxidant molecule, glutathione (GSH) [28
]. Exogenous GSH or L
-cysteine is unabsorbable or unstable outside the cell, respectively. NAC is a representative antioxidant molecule to supply intracellular GSH. NAC has multiple pharmacological effects on osteoblast lineage cells in association with antioxidant capabilities [24
]. NAC is proven to assist bone regeneration on an implant biomaterial by preventing wound infection [29
] and improving the biomaterial cytocompatibility [30
]. Moreover, NAC demonstrated the ability to function as an osteogenic-enhancing molecule, not to induce the differentiation of BMSCs into osteoblast progenitor cells but to promote the osteogenic differentiation of osteoblast-like cells [32
]. Promotion of osteogenic differentiation by NAC may be associated with an increase of cellular GSH through uptake of NAC [33
]. Moreover, preconditioning of naive BMSCs with NAC has been demonstrated to enhance bone regeneration after the local transplantation of autologous BMSCs to a massive femur defect [34
]. The underlying mechanism was to keep high cellular GSH levels in BMSCs long enough to prevent apoptosis and senescence under oxidative condition during acute inflammation phase. Notably, the surviving BMSCs after local transplantation into a bone defect were found near the newly formed bone tissue but not within osteocyte lacunae [34
]. This observation suggests that preconditioning with NAC protected transplanted BMSCs from oxidative stress after local transplantation and that the locally transplanted naive BMSCs were involved in bone regeneration through modulation of the regenerative microenvironment rather than differentiation into osteoblasts.
NAC’s pharmacological effects on BMSCs and osteoblast-like cells raise intriguing questions about local cell transplantation for bone regeneration. The first question is whether preconditioning with NAC is effective for bone regeneration with local transplantation of osteoblasts. The second one is whether the transplanted osteoblasts are involved in bone regeneration as bone-forming cells. NAC improves viability and differentiation of osteoblast-like cells on various bone biomaterials such as the polymethyl-methacrylate resin [30
] and calcium phosphate-based bone substitutes [31
], and collagen matrix scaffolds [37
] by alleviating oxidative stress. It has been hypothesized that preconditioning osteoblasts with NAC prevents suppression of cell viability and differentiation under oxidative stress and promotes bone regeneration after local transplantation in a bone defect by activating bone-forming abilities of the transplanted osteoblasts. The purposes of this study were to examine the effects of incubating osteoblast-like cells with NAC on apoptosis and suppression of osteogenic differentiation induced by oxidative stress and to determine whether preconditioning osteoblast-like cells with NAC enhances bone regeneration in local cell transplantation in a critical-size bone defect. The histological fate of transplanted osteoblast-like cells is also discussed.
This study employed nonclonal BMSCs, which contain stem cells as a subpopulation and are generally used as MSCs for therapeutic purpose [1
]. The cell population may have remained heterogeneous, but it is known that the supplementation of dexamethasone, beta-glycerophosphate, and ascorbic acid into the culture media enhances the osteoblastic differentiation of rat BMSCs [39
]. As shown in Figure 6
, the rat BMSCs grown in the ODM for 2 weeks expressed signatures for osteoblastic differentiation, regardless of NAC preincubation. Hence, nonclonal BMSCs with osteogenic induction were regarded as osteoblast-like cells in the present study.
Our previous study showed that 5 mM NAC enhanced the cellular antioxidant capability in the rat osteoblastic cell culture, but 10 mM NAC reduced cell attachment [32
]. The attached cell density on day 1 in osteoblastic cultures peaked by preconditioning with 5 mM NAC for 3, 12, or 24 h (Figure 1
A). Preconditioning with 5 mM NAC for 3 h kept polygonal cell shapes that was typically observed in cultured osteoblast-like cells [40
] as observed on the cells without NAC preconditioning on day 1; whereas, preconditioning for 6 h changed the cell morphology (Figure 1
B). In addition, total GSH was the highest in the cells pretreated with 5 mM NAC for 3 h and decreased with the further incubation time (Figure 1
C). Therefore, 5 mM and 3 h were set as the NAC preincubation concentration and time, respectively, for osteoblastic cell cultures in the present study. NAC preincubation in such conditions alleviated induction of apoptosis and reduction of cell attachment under exposure to oxidative stress (Figure 2
and Figure 3
). Osteoblast-like cells under exposure to oxidative stress were compromised in cellular redox balance, which was demonstrated by a reduction of total GSH and elevation of cellular ROS. NAC preincubation did not prevent reduction of total GSH but did elevate the cellular ROS levels under exposure to oxidative stress (Figure 3
). This indicated that NAC preincubation prevented a redox imbalance in osteoblast-like cells by reinforcing cellular antioxidant capability to a level high enough to resist contiguous exposure to oxidative stress.
MSCs are known to accumulate in lung tissue after systemic infusion through intravenous injection despite the homing capability to injured tissues [41
]. Likewise, systematically-injected osteoblast-like cells are not located in a bone cavity but distributed in viscera such as lung or liver [18
]. Various cell delivery systems and tissue-engineering technologies based on local cell transplantation have been developed for bone regeneration [2
]. However, oxidative stress is unavoidable for transplanted cells in any implantation procedures, impairing the viability of the transplanted cells and regenerative outcomes [6
]. Preincubation of osteoblast-like cells with 5 mM NAC for 3 h restored osteogenic differentiation suppressed by the exposure to oxidative stress, although cellular proliferation did not improve (Figure 4
and Figure 5
). Under exposure to oxidative stress, NAC preincubation not only improved reductions in the areas of ALP activity and Von Kossa-positive mineralizing nodules but also prevented downregulation of bone matrix-related gene expression, which was not affected by the numbers of attached cells. In addition, osteoblast-like cells preincubated with 5 mM NAC for 3 h had the highest expressions in all osteogenic differentiation markers, such as ALP activity, bone matrix-related genes, and matrix mineralization under growth culture conditions without oxidative stress (Figure 6
). Those observations indicate that NAC preincubation with optimal conditions promoted osteogenic differentiation and enhanced antioxidant capability in osteoblast-like cells.
Our previous study indicated that continuous co-incubation with 5 mM NAC did not induce differentiation of MSCs into osteoblast progenitor cells but promoted differentiation of osteoblast-like cells [32
]. The present study indicated that a single treatment of NAC preincubation influenced osteoblast characteristics for several days after NAC removal while promoting osteogenic differentiation. Although it remains unclear how NAC promotes differentiation of osteoblast-like cells, indirect involvement of NAC through GSH synthesis has been suggested [33
]. Ascorbic acid, which is a representative antioxidant, is known to promote osteogenic differentiation through activation of nuclear factor erythroid 2-related factor (Nrf), a redox signaling pathway [42
]. Nrf proteins are transcriptional factors that not only regulate gene expressions of antioxidant molecules and enzymes in response to cellular ROS [43
] but also bind to the antioxidant response sequence of ocn
]. Exogenous GSH synthesis by NAC preincubation may affect redox signaling pathways as a result of regulation of cellular redox balance. Preincubation with 5 mM NAC for 1 h did not substantially increase total GSH in osteoblast-like cells but promoted osteogenic differentiation (Figure 1
C and Figure 6
). This suggests that the effects of NAC on osteogenic differentiation are independent of GSH synthesis. NAC has the potential to activate the extracellular signal-regulated kinase-mitogen-activated protein kinase (ERK-MAPK) [45
], which also consists of redox-sensitive molecules [46
]. Recently, a biochemical pathway originating from NAC for intracellular production of hydrogen sulfide [47
], which is a strong reductant known to control the osteoblastic function through the ERK-MAPK pathway [48
], has been reported. Influences of cellular redox status and the relative molecules on osteogenic differentiation would be of great interest for future research.
In the present study, the effects of NAC as a cell preconditioning agent on bone regeneration were evaluated in an autologous local transplantation model in a critical-size defect in a rat femur. Autologous local transplantation of osteoblast-like cells formed mineralized structures in the defect to some extent, but the structures were weak and did not cover the entire cortical defect (Figure 7
B). By contrast, local transplants of osteoblast-like cells preincubated with NAC yielded nearly complete defect healing in the form of a thick, compact, and contiguous mineralized structure. The mineralized structure consisted of mature lamellar bone tissue with osteoclastic activity (Figure 8
A–D). Moreover, fluorescent signals meaning the transplanted cells and their daughter cells were detected within osteocytic lacunae in the newly formed bone (Figure 8
E,F,H). Those observations indicate that transplanted osteoblast-like cells pretreated with NAC are involved in the formation of the normal bone tissue with homeostasis as bone-forming cells. By contrast, in the previous study using naive BMSCs in local transplants, transplanted cells were localized in the fibrous tissue adjacent to the newly formed bone tissue but not seen in osteocytic lacunae [34
]. BMSCs pretreated with NAC enhanced bone regeneration as immune regulatory cells with resistance to oxidative stress-mediated apoptosis and senescence during acute inflammation [34
]. NAC preconditioning markedly reinforces resistance to oxidative stress-mediated apoptosis or senescence and enhances immune regulatory function and bone-forming capability in local transplants of BMSCs and osteoblast-like cells, respectively (Figure 9
Preconditioning BMSCs or osteoblast-like cells with NAC brought about an intriguing suggestion that the osteogenic differentiation state of MSCs before transplantation determines the fate of the transplanted cells in local tissue. However, there were some limitations in the present study. First, the terminal fate of transplanted cells was not completely elucidated because of the short observation period of 3 weeks after transplantation. In addition, the characteristics of transplanted cells may not be uniform because nonclonal BMSCs without cell sorting were used. In the present study, many surviving transplanted cells were found near the host osteoblast-like cells on the surface of the newly formed bone as well (Figure 8
E–G). It remained unclear whether those surviving transplanted cells are involved in direct bone formation or modulation of the surrounded cells. Long-term follow-up of the sorted transplanted cells would clarify the role and terminal fate of transplanted cells, depending on the osteogenic differentiation state. Cell preconditioning techniques with NAC may advance transplantation technology and help clarify biological questions in stem cell medicine.
4. Material and Methods
All animal experiments in the present study was performed according to a protocol approved by the University of California at Los Angeles Chancellor’s Animal Research Committee and the Institutional Laboratory Animal Care and Use Committee of Tohoku University (protocol no. 2016-062, approved on 23 September 2016).
4.1. Reagent Preparation and Application
NAC (Sigma-Aldrich, St. Louis, MO, USA) was prepared as a 1 M working solution by dissolving in 1 M 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (Sigma-Aldrich) buffer at pH 7.2. A final NAC concentration for cell conditioning was set as 1 or 5 mM by adding 1 or 5 μL of NAC working solution to 1 mL of cell suspension, respectively. H2O2 (Wako Pure Chemical, Osaka, Japan) was adjusted to 5 mM as a working solution with sterilized ultrapure water. The working solution of H2O2 was used for experiments immediately after preparation. A final concentration of H2O2 for cell culture was set at 50 μM by adding 10 μL of H2O2 working solution into 1 mL of culture media.
4.2. Osteoblastic Cell Culture
BMSCs were isolated from femurs of 8-week-old male SD rats according to the method previously described [49
]. Briefly, the bone marrow tissue was flushed out from femoral cavity. The isolated BMSCs were cultured in an ODM consisting of alpha modification of eagle’s minimum essential medium (α-MEM: GIBCO®
, no nucleosides; Thermo Fisher Scientific, MA., USA), supplemented with 10% fetal bovine serum (GibcoTM
FBS 10082147, Thermo Fisher Scientific), 10−8
M dexamethasone, 10 mM Na-beta-glycerophosphate, 50 μg/mL ascorbic acid, 100 U of penicillin, and 100 μg/mL streptomycin (Wako Pure Chemical) at 37 °C under an atmosphere of 5% CO2
. The cell culture underwent passages two or three times in an ODM for 2 weeks. At 80% confluence, cells were harvested with 0.25% trypsin/1 mM EDTA and then underwent NAC preincubation.
The cells were suspended in a 15 mL conical polypropylene tube (TrueLine TR2001, Nippon Genetics Co.,Ltd., Tokyo, Japan) with a GM consisting of α-MEM, 10% fetal bovine serum, 100 U of penicillin, and 100 μg/mL streptomycin. The cell suspensions were processed according to each protocol of the following three experiments.
In a culture experiment to determine the concentration and treatment time of NAC for osteoblast-like cells, the suspended cells were preincubated with and without 1 or 5 mM NAC. NAC was added into the cell suspension so that co-incubation time with NAC was 1, 3, 6, 12, or 24 h in the 24 h preincubation. After preincubation, the cells were re-suspended into a NAC-free GM and seeded in 12-well culture plates at a cell density of 1.5 × 104 cells/cm2.
In a culture experiment to determine the effect of NAC preconditioning on oxidative stress, the cells suspended with or without 5 mM NAC were preincubated for 3 h. After preincubation, the cells were re-suspended into a NAC-free ODM and seeded in 12-well culture plates at a cell density of 3.0 × 104 cells/cm2 with and without H2O2 addition.
In a culture experiment to determine the effect of NAC preconditioning on osteoblastic differentiation of osteoblast-like cells, the suspended cells were preincubated with and without 5 mM NAC. NAC was added into the cell suspension so that co-incubation time with NAC was 1, 3, or 6 h in the 6 h preincubation. After preincubation, the cells were re-suspended into a NAC-free ODM without dexamethasone and seeded in 12-well culture plates at a cell density of 1.5 × 104 cells/cm2.
The culture medium corresponding to each experiment was renewed at intervals of 3 days.
4.3. Cell Attachment, Cytomorphometry, and Proliferation Assay
The numbers of attached cells were evaluated with cell density measurements and a WST-1 assay. For cell density measurements, the attached cells were gently rinsed twice with phosphate buffered saline (PBS) on days 2 and 5 after seeding and then detached with 300 μL of 0.25% trypsin/1 mM EDTA-4Na for 15 min at 37 °C. A hematocytometer (Bright-Line, Hausser Scientific, PA, USA) was used to count the number of collected cells. The substrates were examined under a microscope to confirm no remaining cells. For the WST-1 assay, the culture medium was replaced on day 1 after seeding with a fresh medium containing a WST-1 reagent (Roche Diagnostics, Indianapolis, IN, USA). Then, the cells were incubated for 3 h at 37 °C under an atmosphere of 5% CO2. After gentle shaking, the optical density (OD) for formazan in the supernatant was measured at 450 nm in the microplate reader, which had a linear relationship with the number of cells attached on substrates.
Cytomorphometry was performed on phase contract microscopic images in the day 1 culture using an image analyzer (ImageJ, NIH, ML, USA). For each culture, 25 cells were randomly selected and analyzed for area, perimeter, and Feret diameter.
Cellular proliferation activity was evaluated with BrdU incorporation assay (Sigma-Aldrich). On day 2, the culture medium with H2O2 was renewed into a fresh GM containing 100 mM BrdU solution and then the cells were incubated for additional 10 h. After DNA was denatured, cultures were incubated with anti-BrdU conjugated with peroxidase. Then, tetramethylbenzidine was reacted for color development. Absorbance was measured at 370 nm in the microplate reader.
4.4. Flow Cytometry for Apoptosis Detection
With exposure to H2O2 for 24 h, apoptotic and necrotic appearances were evaluated using a flow cytometry with annexin V-FITC and propidium iodide (PI) staining (Annexin V-FITC Kit: Beckman Coulter, CA, USA). This staining is based on the principles that annexin V binds to phosphatidylserine expressed on the surface of apoptotic cells and that membrane-impermeable PI is incorporated into the DNA of necrotic cells. Both floating and attached cells were collected into a tube. The cell suspension were treated with 25 μL of annexin V-FITC and 12.5 μL of PI (6.25 μg/mL) for 10 min in the dark on ice. After being filtered with a cell strainer, the cell suspension was analyzed using a FACS Aria II system (Becton Dickenson, Franklin Lakes, NJ, USA).
4.5. GSH Detection Assay
Cells were washed with PBS and incubated with 600 μL of 10 mM hydrochloric acid on each well. The cell lysate was obtained by repeating freeze–thaw cycles two times. The cell lysate was then mixed with 5% sulfosalicylic acid. After its centrifugation at 8000 g for 10 min, the supernatant was collected and dispensed in 40-μL batches into 96-well plates for the assay with a GSH detection kit (Total Glutathione Quantification Kit: Dojindo Molecular Technologies, Inc., Rockville, MD, USA). Briefly, after adding a buffer solution, the plate was incubated at 37 °C for 60 min. Then, a 5,5-dithiobis (2-nitrobenzoic acid) (DTNB) substrate and a GSH reductase were added to each well, and the plate was incubated at 37 °C for 10 min for chromogenic reaction of DTNB in the presence of GSH. The OD of DTNB in each well was measured using the microplate reader at 405 nm. Total GSH in each culture was calculated using a calibration curve for the standard GSH solution. To evaluate total GSH per unit cell in cell suspensions co-incubated with 5 mM NAC for 0, 3, 6, or 12 h in the 12 h preincubation, the values of total GSH were divided by cell numbers in the corresponding duplicated cell suspensions. To evaluate the total GSH in the cell culture after exposure to H2O2, the floating cells were also collected and combined with the adherent cells into a cells lysate.
4.6. Intracellular ROS Level
On day 2 after exposure to H2O2, cells were washed with PBS and incubated in 1000 μL of PBS containing 10 μM H2DCFDA (Thermo Fisher Scientific) for 15 min at 37 °C in a 5% CO2 atmosphere. H2DCFDA is membrane-permeable and intracellularly changes into fluorescent dichlorofluorescin diacetate (DCFDA) as a result of oxidization by ROS. The fluorescence intensity of DCFDA was measured using a multimode microplate reader (excitation: 490 nm; emission: 510–570 nm). The intracellular ROS level was determined according to the fluorescence intensity values, which were normalized against the amount of cells in a duplicate culture measured with a hematocytometer.
4.7. Gene Expression Analysis
Expressions for bone matrix-related genes were analyzed using RT-PCR on day 10. Total RNA in the cultures was extracted using the TRIzol reagent (Thermo Fisher Scientific) and a purification column (RNeasy, Qiagen, CA, USA). Following DNAse I (Ambion™ DNase I, Thermo Fisher Scientific) treatment, reverse transcription of 0.5 μg total RNA was performed using MMLV reverse transcriptase (Clontech, CA, USA) in the presence of oligo(dT) primer (Clontech). PCR on first-strand DNA was performed using Taq DNA polymerase (TaKaRa Ex Taq, Takara Bio, Shiga, Japan) to detect col1a1, opn, and ocn mRNA. Glyceraldehyde-3-phosphate dehydrogenase (gapdh) was employed as a house-keeping gene. The forward and backward primers (Sigma-Aldrich) were col1a1: 5′-GGCAACAGTCGATTCACC-3′ and 5′-AGGGCCAATGTCCATTCC-3′, opn: 5′-GATTATAGTGACACAGAC-3′ and 5′-AGCAGGAATACTAACTGC-3′, ocn: 5′-GTCCCACACAGCAACTCG-3′ and 5′-CCAAAGCTGAAGCTGCCG-3′, and gapdh: 5′-TGAAGGTCGGTGTCAACGGATTTGGC-3′ and 5′-CATGTAGGCCATGAGGTCCACCAC-3′. The PCR products were visualized on a 1.5% agarose gel by ethidium bromide staining under ultraviolet (UV) light.
4.8. Alkaline Phosphatase Activity
Alkaline phosphatase (ALP) activity of the culture was evaluated with azo dye staining. On day 3, cells were washed twice with Hanks’ solution and incubated with 120 mM Tris buffer (pH 8.4) containing 0.9 mM naphthol AS-MX phosphate and 1.8 mM fast red TR for 30 min at 37 °C. The ALP-positive area (%) on the stained images was calculated as (stained area/total dish area) × 100 using Image J.
4.9. Matrix Mineralization Assay
Matrix mineralization of the culture on days 14 and 21 was examined by quantification of total calcium deposition and Von Kossa staining to visualize the mineralized area in the culture, respectively.
For quantification of total calcium deposition, cells were washed with PBS and incubated overnight in 1 mL of 0.5 M HCl solution with gentle shaking. The solution containing the eluted calcium ions was mixed in an alkaline medium with o-cresolphthalein complexone (Calcium Assay Kit, Cayman Chemical Company, MI, USA), which reacts with calcium ions to form a purple cresolphthalein complexone complex. After gentle shaking, the OD for a cresolphthalein complexone was measured at 570 nm in the microplate reader. The amount of calcium deposition in each culture was calculated using a calibration curve for the standard calcium solution.
For Von Kossa staining, cells were fixed using a solution of 50% ethanol/18% formaldehyde solution for 30 min. The cultures were then incubated with 5% silver nitrate under UV light for 30 min. Finally, the cultures were washed twice with double-distilled H2O and incubated with 5% sodium thiosulfate solution for 2–5 min. The Von Kossa positive area (%), defined as (stained area/total dish area) × 100, was measured using an image analyzer.
4.10. Autologous Local Transplantation in A Massive Rat Femur Bone Defect
Autologous local transplantation in critical-size defects in rat femurs was performed based on the protocol in the previous study [34
]. Eleven-week-old male SD rats were anesthetized with 2% isoflurane. A small unicortical hole penetrating to the bone marrow space was drilled 5 mm from the distal end of the epiphyseal region of the right femur. Approximately 500 μL of bone marrow was aspirated with a sterilized syringe and immediately suspended in the ODM. After collection of bone marrow, the muscle and the skin in the open wounds were sutured with 4-0 vicryl and 3-0 silk, respectively.
The harvested bone marrow tissue was plated on a 10-cm cell culture dish and incubated at 37 °C in a 5% CO2
atmosphere. After 24 h, the nonadherent cells were removed by washing with PBS. The culture medium was renewed at intervals of 3 days. After 14 days (when the cells reached 80% confluence), the cells were labeled with 1 nM fluorescent organic dot cell tracer (Long Term Cell Tracer 500 Cat. # P710G; 101 Bio.com
, CA, USA) for 12 h in suspension. This cell tracer is known to be non-cytotoxic and is transmitted to daughter cells. Subsequently, cells were treated with 5 mM NAC for 3 h and then suspended again in a NAC-free GM. A bovine tendon-derived type I collagen sponge (Collaplug, Integra LifeSciences Corp, NJ, USA) [50
] was used as a vehicle. The cell suspension was added into the inside of each sponge and incubated overnight at 37°C in a 5% CO2
The left femur of the rat was subjected to a second auto-transplantation surgery. The rats were 13 weeks old at the time of the secondary surgery. After exposure of the left femur bone, a large rectangular segmental resection (5 × 5 × 2.5 mm), which was larger than the critical-sized defect in the rat femur previously reported [32
], was made under irrigation in the center part of the cortical bone of the femur. The distal end of the defect was positioned 10 mm from the distal end of the epiphyseal region of the femur bone. Collagen sponges containing autologous osteoblast-like cells with and without NAC treatment were implanted into the segmental defect. The implantation site was wrapped with an absorbable collagen membrane (Koken Tissue Guide; Olympus Terumo Biomaterial, Tokyo, Japan). The femur bone was prevented from a fracture using a titanium splint and a glass fiber-containing hydraulic polyurethane resin-based dressing material. The muscle and the skin were sutured with 4-0 vicryl and 3-0 silk, respectively.
4.11. Micro-Computed Tomography Analysis
Three weeks after surgery, the left femur was excised and fixed in 10% neutral buffered formalin (Wako Pure Chemical) at 4 °C for 1 week. All splinting materials were removed during the fixation. Bone volume and mineral density of the regenerated bone at the site of the autologous cell implantation were evaluated using a ScanXmate-E090 device for three-dimensional micro X-ray computed tomography (micro-CT) (μCT 40, Scanco Medical AG, Bassersdorf, Switzerland) with an isotropic resolution of 8 μm. The femur specimens were X-rayed at an energy level of 70 kVp and a current of 114 μA. Grayscale images were processed using a Gaussian low-pass noise filter and threshold algorithms to distinguish between mineralized bone and the background. The area of interest for analysis was set on the combined area of the cortical and bone marrow space regions, which was defined as the region surrounded by connecting external basic lamellar points of both sides of the defect edges and the corresponding points on the opposite internal basic lamellar surface. The specific thresholds for bone tissue were determined by superimposing segmented images over the original grayscale images. A quantitative assessment was made of the following three dimensional parameters within the area of interest among groups: BV/TV (%), Tb. N (1/mm), Tb. Th (μm), and Tb. Sp (μm).
4.12. Histological Analysis
After micro-CT scanning, the samples were delipidated in ethanol and acetone and decalcified in a 10% EDTA disodium salt (EDTA 2Na) at 4 °C for 3 weeks. After dehydration in ethanol series, acetone, and xylene, samples were embedded in paraffin. Paraffin sections (3–5 μm) were stained with hematoxylin and eosin (H&E) or TRAP staining for morphological observation to check bone tissue structure and localization of the transplanted cells. For TRAP staining, the deparaffined sections were treated with a mixture of a tartaric acid solution and acid phosphatase substrates for 60 min at room temperature. Hematoxylin was used for counterstaining. Tissue morphology was observed in H&E and TRAP-stained sections under the bright-field setting on an all-in-one fluorescent microscope (BZ-9000, Keyence). The distribution of labeled transplanted cells was observed in the corresponding region of H&E-stained sections under the fluorescence setting with a 4′,6-diamidino-2-phenylindole filter (excitation: 400 nm; emission: 510 nm).
4.13. Statistical Analysis
One-way analysis of variance was used to assess the differences among multiple experimental groups. When appropriate, Dunnett’s test and Tukey’s HSD test were used as a post-hoc test. Student’s or Welch’s t-test was used to compare the two groups, and p < 0.05 was considered statistically significant. A statistical analysis was performed using IBM SPSS Statistics 21 statistical software (IBM Japan, Ltd., Tokyo, Japan).