Objectives: Through the combination of cell-based therapies and 3-dimensional scaffolds, bone tissue engineering presents promising options for treatment of bone defects.
Materials and Methods: In this study, we used an animal model to investigate the regenerative potential of hydroxyapatite scaffolds treated with mesenchymal stem cells-conditioned medium on bone tissue engineering (bone defect repair).
Results: Stem cells were cultured and identified by flow cytometry, which confirmed the high expression of mesenchymal markers (CD146, CD90, CD105) and minimal expression of the CD31 and CD45 hematopoietic markers. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay revealed that 3T3 cells in scaffolds treated with a conditioned medium were more viable than untreated controls. Scratch test revealed that cell migration in the treated scaffolds was significantly improved. Histological staining and histomorphometric analysis in a rat model with calvaria defects verified that the treated scaffolds significantly promoted bone regeneration.
Conclusions: These findings highlight the potential of these hybrid scaffolds as effective and biocompatible strategy to advance bone tissue regeneration in clinical applications.

Key words : Biocompatible strategy, Cranial defect model, Mesenchymal markers, Tissue regeneration

Introduction

Large bone defects caused by trauma, infections, or tumors pose significant treatment challenges because of the limited capacity of bone regeneration.^1,2 Traditional therapies such as autografts and allog-rafts are associated with problems such as donor site complications, immune rejection, and inadequate integration.^3,4

Biomaterial scaffolds such as hydroxyapatite (HA) provide osteoconductive matrices that mimic bone mineral, but their acellular versions lack sufficient bone-inducing signals to fully regenerate large defects.⁵ Hydroxyapatite has garnered a lot of interest due to its properties such as good biocompatibility, bioactivity, good bone binding, and lack of immune system stimulation.^6,7 Bone tissue engineering, by combining biocompatible scaffolds and bioactive agents, offers a promising solution.

Mesenchymal stem cell-conditioned medium (MSC-CM) contains a combination of cytokines, growth factors, and extracellular vesicles that collec-tively promote osteoblast proliferation, migration, and matrix mineralization.⁸ Mesenchymal stem cells (MSCs) are involved in a wide range of biological processes, such as cell migration, proliferation, differentiation, anti-inflammation, and activation or inhibition of signaling pathways^8,9 and also support angiogenesis, modify the habitat, and influence the immune system for tissue repair.^8,9 Researchers are moving toward a cell-free therapeutic strategy by using conditioned medium to overcome the challenges associated with cell therapy.

Few studies are available on the use of MSC-CM for the repair of traumatic injuries.10,11 Recent in vitro and in vivo studies have shown that the combination of MSC-CM with HA scaffolds significantly accele-rates new bone formation versus the scaffold alone.^12,13

Therefore, the purpose of this study was to determine the efficacy of simultaneous transplant of HA scaffolds treated with MSC-CM on the repair of bone lesions (bone tissue engineering) in a rat model. The results of this study may contribute to the development of advanced biomaterial-based therapies for bone tissue engineering and provide a synergistic approach to enhance the performance of scaffolds through active conditioned medium.

Materials and Methods

Study design
This study was designed to evaluate the efficacy of MSC-CM-treated HA scaffolds in bone regeneration. First, we used flow cytometry to identify mesenchymal markers. We then prepared HA scaffolds and treated these with MSC-CM. We performed in vitro experiments, including 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay (MTT assay) for assessment of cell viability and scratch assay for assessment of cell migration, to evaluate the biological effects of this combination. Finally, we performed an in vivo study with a cranial defect model in Sprague-Dawley Wistar rats, in which MSC-CM-treated HA scaffolds were implanted and analyzed after 4 weeks with hematoxylin and eosin (HE) and Masson trichrome staining.

Mesenchymal stem cell culture and identification
The MSCs were obtained from the National Center for Genetic and Biological Resources of Iran. Specifically, bone marrow-derived MSCs were used in this study. All cell culture steps were performed under biosafety level BSL-2 conditions to ensure safety and sterility. Cells were thawed and passaged and then cultured in large flasks for subsequent experiments. The cells were grown in DMEM medium at 37 °C, 5% CO2, and 95% humidity with 10% fetal bovine serum (FBS), 100 U/mL of penicillin, and 100 μg/mL of streptomycin.14 After the third passage, these were used for differentiation characteristics and cell surface markers.¹⁴

Immunophenotype of mesenchymal stem cells
Immunophenotype of cells in terms of expression of MSC markers was examined according to the criteria of the International Society for Cellular Therapy.¹⁵ By this method, endometrial MSCs can be identified by the expression of CD146, CD90, and CD105 as mesenchymal markers and the lack of expression of CD34, CD45, and CD31 as markers of hematopoietic and endothelial stem cells.¹⁵ For this purpose, the cells in the third passage were first washed with phosphate-buffered saline (PBS), separated from the flask surface by trypsin-EDTA enzymatic treatment, and then centrifuged for 5 minutes at 2000 rpm. After supernatant was discarded, cell counting was performed, after which the sample was centrifuged again. Cells were then washed with PBS at pH 4.7. For each marker, the protocol requires that 10⁶ cells be placed in PBS in separate tubes. We added 3 μL of fluorochrome antibodies (phycoerythrin, fluorescein isothiocyanate, or peridinin chlorophyll protein) to the cells, which were then incubated for 20 minutes at room temperature in the dark. For the next step, the cell suspension was centrifuged with PBS for 10 minutes at 2000 rpm. We then added 1% formalin fixing buffer to the cells, after which we examined expression of the markers by flow cytometry (FACSCalibur, BD Biosciences); we analyzed results with flow cytometry data analysis software (FlowJo, TreeStar).

Preparation of conditioned medium from mesenchymal stem cells
After the cells were thawed and passaged, the MSCs were cultured in large flasks for further experiments. Cells were grown in DMEM medium at 37 °C, 5% CO₂, and 95% humidity with 10% FBS, 100 U/mL of penicillin, and 100 μg/mL of streptomycin. After a suitable confluence was achieved (80%), the supernatant of the MSCs was collected and then passed through a 0.22-μm filter. Next, the exosome-saturated granules were implanted into the bone lesion of the rat skull. After 4 weeks, the repair was examined by histology.

Effects of hydroxyapatite scaffolds treated with conditioned medium by colorimetric assay
We used the MTT assay to investigate the nontoxicity of HA scaffolds treated with MSC-CM. The MTT test is typically performed on 3T3 mouse fibroblast cells by direct method. In this way, cells are seeded on MSC-CM-treated HA scaffolds and incubated for 24, 48, and 72 hours, after which the medium is removed at different times and incubated with MTT for 3 hours. In the final step, MTT is removed, and dimethyl sulfoxide is added. The results of each experiment should be repeated at least 3 times.

Effects of hydroxyapatite scaffolds treated with conditioned medium on cell migration using scratch assay
We cultured 3T3 mouse fibroblast cells at an appropriate density in separate 24-well plates with DMEM-F12 medium and 10% FBS to reach a confluence of about 70% to 80%. With 100-μL pipette tips, we gently made a full-length scratch with minimal pressure to the cells. After the scratch was created, the cells were treated with a fresh culture medium with different concentrations of scaffold extract and conditioned media. One group was considered a control. Cell movement and migration into the scratch area were examined at 0, 12, and 24 hours by using a phase-contrast microscope and ×10 magnification. The number of cells that had migrated into the damaged area was counted using image analysis software (ImageJ, open source) and used for quantification. Each experiment was repeated at least 3 times.

Synthesis of hydroxyapatite bone scaffolds
The HA was obtained from the Iranian Tissue Product Company (Regen Allograft), and implant scaffolds were created from the prepared HA powder.¹⁶ To achieve the required viscosity for molding, 10% (wt/vol) polyvinyl alcohol (PVA) was first added to the powder as a binder (0-3 g HA with 4 drops of PVA). A polymer solution with a concentration of 10% (wt/vol) was created by dissolving PVA powder, which has a molecular weight of 72 000 g/mol, in water at 80 °C. To create disk-shaped scaffolds, this mixture was subsequently transferred to a specialized mold. Following pre-paration, to burn out the PVA, the scaffolds were first placed in an electric furnace and heated to 300 °C for 1 hour. For full curing, the temperature was then increased to 1100 °C for 1 hour.

In vivo procedure
All animal experiments were approved by the Ethics Committee of Semnan University of Medical Sciences (approval No. IR.SEMUMS.REC.1399.048). The HA scaffolds were incubated with exosome solution for 12 hours overnight before surgery, after all scaffolds had been sterilized by ultraviolet exposure for 15 minutes on each side. For this study, we used 15 female Sprague-Dawley Wistar rats with an average body weight of 280 g and age of 5 to 6 weeks, which we kept in the animal facility under standard conditions with adequate access to water and food. The rats were randomly divided into 3 groups (5 rats per group), which included (1) a control group with bone defect but without scaffold, (2) an experimental group with the untreated HA-based scaffold (no MSC-CM), and (3) an experimental group with the MSC-CM-treated HA-based scaffold. Rats were anesthetized with xylazine (2-10 mg/kg) and fixed in a stereotaxic apparatus. The surgical site was first shaved and sterilized. Then, trephine holes were created using a burr and circular saw to produce 8-mm critical-sized defects via an anterior-to-posterior approach. The prepared scaffolds were placed in the newly created bone defect in the calvaria. All rats received an antibiotic treatment of 5% enrofloxacin. At 4 weeks after implant, we removed the implanted material along with the surrounding tissues.

For histological analyses, the tissue of interest (cranial bone defect area) was dissected from killed rats, and then HE and Masson trichrome staining experiments were performed. The prepared slides were then examined by fluorescence microscopy (model BX51, Olympus). We used image analysis software (Image-Pro Plus, version 6) to calculate and analyze the number of cells, including fibroblasts, fibrocytes, osteoblasts, and osteons, for histomorp-hometric analysis.

Statistical analyses
Data are presented as mean values with SD from at least 3 independent experiments. We compared multiple groups by 1-way analysis of variance, with pairwise comparisons conducted by using unpaired t tests. P < .05 was considered statistically significant. We used statistical graphing software (Prism 10, GraphPad) for all analyses.

Results

Culture and immunophenotyping of bone marrow mesenchymal stem cells
Bone marrow MSCs were cultured and passaged several times; when the cells reached appropriate confluence (80%), the morphology of the cells was examined using a light microscope. Light microscope images showed that the cells exhibited appropriate morphology, appearing round and spindle-shaped.

The CD146, CD90, and CD105, which are mesenchymal markers, and the CD31 and CD45, which are blood markers (selected for control), were examined to confirm the nature of the cells. After the third passage of the cells, surface markers were examined using flow cytometry. The results of flow cytometry analysis showed that these cells expressed 98.4% of CD90, 46.5% of CD146, and 76.7% of CD105. Furthermore, consistent with criteria defined by the International Society for Cellular Therapy, the cells exhibited minimal expression of hematopoietic stem cell markers CD31 (1.97%) and CD45 (1.67%), which confirmed the absence of hematopoietic contamination and the purity of the MSC population (Figure 1).

Proliferative effects of conditioned medium with hydroxyapatite scaffold on 3T3 cells
To investigate the effect of MSC-CM-treated HA scaffolds on 3T3 fibroblast cells, the viability of cells within the scaffold was examined in the laboratory using the MTT assay. We performed the MTT assay on the 3 groups (MSC-CM-treated HA scaffold group, HA scaffold group without MSC-CM, and the control group), and the results of MTT showed the nontoxicity of cells on MSC-CM-treated HA scaffolds and the proper growth of these cells. In addition, by performing this test and comparing cell viability, we observed that the survival rate of cells on the MSC-CM-treated HA scaffold group significantly increased versus the HA scaffold without MSC-CM group and the control group (Figure 2).

Migratory effects of hydroxyapatite scaffold with conditioned medium on 3T3 cells
Scratch test was performed to investigate the migration of bone marrow stem cells affected by HA scaffold without MSC-CM treatment and HA scaffold treated with MSC-CM. We performed scratch test at 3 time intervals (0, 24, and 48 hours) and on the following 3 groups: MSC-CM=treated HA scaffold group, untreated HA scaffold group, and control group (no HA scaffold) (Figure 3). According to the results obtained from the scratch test, cell migration in the MSC-CM-treated HA scaffold group increased significantly with time versus the untreated HA scaffold group and the control group. Microscopy images of groove filling were also analyzed using Image J software. Results versus the control group were considered significant.

Histological analyses
For histological analyses, rats were killed, and the desired tissue (cranial bone defect area) was cut, after which HE and Masson trichrome staining expe-riments were performed (Figure 4). Prepared slides were then examined by fluorescence microscopy (model BX51, Olympus) with image analysis software (Image-Pro Plus, version 6) used to calculate and analyze the number of cells. A magnification of ×200 was used for cell counting (Figure 4). According to HE staining and Masson trichrome staining, although there was a small amount of incompletely formed new bone at the end of the experiment, the control group samples were filled with fibrous connective tissue. In the MSC-CM-treated HA scaffold group, the amount of new bone was significantly increased versus the control group. In addition, although fibrous connective tissue was still present, it was significantly reduced versus the control group. Histomorphometric analysis showed that area of new bone was significantly different between the treatment group and the control group with total number of osteoblasts-osteocytes and osteons significantly higher in the treatment group than in the control group (Figure 5).

Discussion

Bone is the second most abundant connective tissue in the body and has a Bone grafts or bone substitutes are used to treat bone defects. However, significant limitations (for example, cancer or infection) may affect the presently established treatments; therefore, there is a persistent need for appropriate clinical solutions to replace bone.^17,18 Hence, scaffolds are considered suitable biological substitutes for bone defects, and as such these may improve and accelerate the healing process of the surrounding tissue.

Presently, bone tissue engineering is accomplished with cells and 3-dimensional biodegradable scaffolds to repair damaged bone tissue. Thus, the design and construction of the appropriate scaffold and the use of the cells are of particular importance.

Because cells alone cannot be a suitable substitute for bone tissue, a structure is needed on which the cells can settle and grow. A porous material should therefore be considered as an extracellular matrix or scaffold for cell growth.^19,20 The location of the bone tissue in the body will determine the required characteristics of these scaffolds. In all cases, the scaffolds must be biocompatible and mimic the properties of bone at the site of injury. The properties of the macrostructures and microstructures are very important. These properties not only affect cell survival, growth, reorganization, and signaling, but also affect gene expression and the maintenance of the cellular phenotype. For this reason, 3-dimen-sional structures of bone extracellular matrix biomimetic scaffolds have been applied to meet this need. By incorporation of bioactive mineral compounds, the mechanical and physical properties of bone extracellular matrix can be further simulated, and thereby the biological performance of the resulting biomaterial scaffold can be enhanced.^21,22

Among ceramics, calcium phosphate ceramics have received the most extensive research, with HA being one of the most widely used.²³ When used in bone substitutes, these materials exhibit good biocompatibility and bone conduction.^23,24 Previous studies have shown that, by using ceramics along with MSC-CM, in addition to achieving better mechanical properties, the degree of The conditioned medium is defined as all of the contents of the MSC culture medium that remain free of cell debris and dead cells.^26,27

In this project, we used HA scaffolds because HA has inductive properties and is used as a substitute for cancellous bone. We also investigated HA scaffolds that had been treated with MSC-CM. The mechanisms by which MSC-CM promotes bone regeneration are multifaceted. The MSC-CM contains a rich set of growth factors, including vascular endothelial growth factor, bone morphogenetic protein 2, and transfor-ming growth factor-β, which are critical for bone formation and angiogenesis.^11,28 These factors not only stimulate the proliferation and differentiation of osteogenic cells but also enhance the recruitment of endogenous stem cells to the injury site, which enhan-ces the regenerative response. In addition, MSC-CM has anti-inflammatory effects and, by modulating the immune response, reduces inflam-mation and creates a favorable environment for tissue repair.¹¹ In our study, the significant increase in bone formation in the MSC-CM-treated HA scaffold group versus the other groups demonstrated the strong regenerative capacity of MSC-CM when combined with HA scaffolds.

In our study, after cells were cultured in flasks, the cells formed distinct colonies around single cells. Also, flow cytometry experiments to identify markers of MSCs showed positivity of these cells with CD146, CD90, and CD105 and negativity toward CD31, in ag-reement with the findings of Zhang and colleagues.²⁹

The osteogenic differentiation potential of MSCs was confirmed through in vitro experiments, which indicated the suitability of MSCs for bone rege-neration applications. In addition to the surface marker profile, we confirmed that human bone marrow-derived MSCs have a strong trilineage differentiation capacity. Under osteogenic induction, these cells showed significant Alizarin Red S staining by day 21, whereas adipogenic and chondrogenic environments produced abundant lipid droplets (positive staining with Oil Red O) and sulfated proteoglycans (positive staining with Alcian Blue), respectively. These findings are consistent with established definitions of MSCs^30,31 and emphasize the inherent pluripotency that likely contributed to the osteogenic function observed in our MSC-CM-treated HA hybrid scaffold.

The 3-dimensional cell culture on MSC-CM-treated HA scaffolds, performed in previous studies, showed increased survival rate of cells on this scaffold.^32,33 Such results indicate the suitability of the scaffold for cells. Therefore, the cytotoxicity of the fabricated scaffolds was examined with the MTT assay, which indicated the nontoxicity of the resulting scaffolds. In vitro cell studies thus showed that these scaffolds did not cause any toxicity to the body and were a suitable substrate for cell growth, proliferation, and differentiation. After the initial properties and characteristics of the synthesized scaffolds were established, in vivo studies of these biomaterials were conducted. For this purpose, MSC-CM-treated HA scaffolds and untreated HA scaffolds were placed in the bone defect site in the calvaria of the animal after creating a bone lesion in the rat skull. After 1 month of this process, tissue samples stained with HE showed that bone regeneration is a time-dependent process, with more healing and repair occurring with increasing time. As shown in Figure 4, the results of bone repair and regeneration in the MSC-CM-treated HA scaffold group differed from the control group. In the control group, the defect area was mostly filled by fibrous connective tissue. However, in the treatment group, the defect area was mostly filled by new bone formation. Histomor-phometric analysis showed a significant difference regarding new bone between the treatment group and the control group (Figure 5). In addition, the presence of these scaffolds in the cranial defect of the rat model indicated the formation of bone tissue within 1 month.

Evaluation of in vivo bone formation showed that bone regeneration is a time-dependent process. Histological studies revealed the strength of bone formation. The staining results showed that bone regeneration occurred in the groups with MSC-CM-treated HA scaffolds. Our study demonstrated that stem cells offer numerous possibilities for the development of novel biological clinical therapies aimed at bone tissue regeneration.

Although we demonstrated the efficacy of combining HA scaffolds with MSC-CM for bone regeneration, we note several limitations. First, the long-term stability and degradation rate of HA scaffolds require further investigation (long-term degradation kinetics in vivo >12 weeks) to ensure that sustained support for bone formation is provided during extended periods. Second, although our in vivo model provided valuable insights, the immune response and regeneration outcomes may differ in larger animal models or humans, and so further validation is required. Comparative immunohistoc-hemistry across species is essential. Furthermore, the scalability of MSC-CM production for clinical applica-tions is challenging, because large-scale production requires effort to maintain the consistency and robustness of the conditioned medium. Future studies should focus on optimization of MSC culture conditions to enhance the production of osteogenic factors and development of standardized protocols for MSC-CM preparation to facilitate translation into clinical practice.

Conclusions

This study demonstrated that HA scaffolds treated with MSC-CM significantly improved bone rege-neration in a rat cranial defect model. The HA scaffolds, which provide a biocompatible and osteogenic framework, in combination with MSC-CM, which is rich in growth factors and regenerative molecules, significantly enhanced cell viability, migration, and new bone formation versus untreated scaffolds or controls. These findings demonstrated the potential of this combined approach to overcome the limitations of current bone graft materials and offer a promising strategy for clinical applications in regenerative medicine. Future research should focus on optimization of this system for human use, including scalability and long-term stability.

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