Bone regeneration using stem cell spheroids within 3D-printed scaffolds in a rabbit radial defect model

Bone regeneration using stem cell spheroids within 3D-printed scaffolds in a rabbit radial defect model | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Bone regeneration using stem cell spheroids within 3D-printed scaffolds in a rabbit radial defect model Yangwon Chae, Kwangsik Jang, Sol Lee, Yong-hun Kim, Songwan Jin, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6291864/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 16 Dec, 2025 Read the published version in Scientific Reports → Version 1 posted 11 You are reading this latest preprint version Abstract Bone tissue is generally resilient and can self-heal, but critical-size defects (CSDs) with complex geometries cannot be repaired without clinical intervention. Customized scaffolds developed using three-dimensional (3D) printing techniques can effectively repair complex-shaped CSDs. Adipose-derived stem cells (ADSCs), a type of mesenchymal stem cell (MSC), can differentiate into osteoblasts and exhibit osteoinductive properties. However, ADSC-single cells fabricated via two-dimensional (2D) monolayer cultures have limitations in maintaining cell survival and function over time. Unlike 2D monolayer cultures, ADSC-spheroids fabricated via 3D spheroid cultures can overcome this limitation by increasing the survival of ADSCs and enhancing their in vivo osteogenic capacity. This study aimed to evaluate the potential of a synergistic strategy of ADSC-spheroids within a 3D-printed scaffold made of polycaprolactone/hydroxyapatite (PCL/HA) in bone regeneration. In vitro experiments demonstrated that ADSC-spheroids promoted mineralization in 3D-printed scaffolds. Radiographs and histological analysis performed at eight weeks post-implantation in in vivo experiments using a rabbit radial defect model showed successful bone regeneration in the group containing ADSC-spheroids within the PCL/HA scaffold. These results suggest that the synergistic strategy of incorporating ADSC-spheroids into 3D-printed PCL/HA scaffolds shows promise for clinical applications in treating complex CSDs. Biological sciences/Biotechnology/Biomaterials/Biomedical materials Biological sciences/Biotechnology/Biomaterials/Tissues critical-size defect bone tissue engineering spheroids synergic strategy 3D-printed scaffold bone regeneration Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Bone defects resulting from inflammation, trauma, tumors, or congenital anomalies present significant challenges to bone regeneration [ 1 ]. While bone tissue generally exhibits resilience and self-healing abilities, critical-size defects (CSDs) necessitate therapeutic intervention for proper repair [ 2 , 3 ]. Autologous bone grafts are considered the gold standard for treating these defects due to their favorable osteoinductive, osteoconductive, and cellular properties [ 4 ]. However, this approach has limitations including donor site morbidity, long surgical procedures, and limited availability [ 5 ]. Although alternative bone grafting materials such as allografts, xenografts, and alloplasts can address some of these issues, they have the disadvantage of reduced osteogenic capacity due to their lack of osteoinductive potential. Furthermore, these particle-type bone grafts are less likely to maintain their shape in CSDs, making it difficult to provide a structural framework for new bone formation [ 6 , 7 ]. Stem cell-based therapies are emerging as promising solutions in bone tissue engineering (BTE) to enhance osteoinductive potential [ 8 ]. Mesenchymal stem cells (MSCs) are multipotent cells in various connective tissues throughout the body including the fetal liver, umbilical cord blood, adipose tissue, and adult bone marrow [ 9 – 12 ]. Adipose-derived stem cells (ADSCs), a type of MSC derived from adipose tissue, can differentiate into osteoblasts and exhibit osteoinductive properties [ 13 ]. Their osteoinductive potential is attributed to their high proliferation rates, low immunogenicity, and capacity to differentiate into multiple cell types [ 9 , 14 ]. While some studies have demonstrated the effectiveness of ADSCs in promoting bone regeneration, ADSC-single cells fabricated via two-dimensional (2D) monolayer cultures have limitations in maintaining cell survival and function over time [ 15 ]. These limitations may arise from the unphysiological microenvironment created by traditional 2D monolayer culture techniques [ 16 ]. Recent advances in BTE have revealed the benefits of ADSC-spheroids fabricated via three-dimensional (3D) spheroid culture techniques [ 17 ]. This approach allows for direct cell-to-cell interactions, facilitating the formation of spheroids [ 18 ]. Unlike 2D monolayer cultures, 3D spheroid cultures better replicate the in vivo environment, enhancing cellular interactions, extracellular matrix (ECM) production, and overall tissue organization [ 19 ]. Moreover, the unique geometry of 3D spheroid cultures can induce anti-inflammatory, anti-apoptotic, and pro-angiogenic effects [ 20 – 22 ]. These characteristics enhance the osteoinductive potential of ADSCs in pathological bone repair, particularly in addressing challenges such as avascularity and hypoxia in large defects [ 23 ]. These improvements in ADSC-spheroid technology could significantly benefit the treatment of CSDs. Several studies [ 24 – 26 ] have shown that spheroids of ADSCs effectively promote bone growth. Scaffolds serve as 3D structures that provide a transient environment for cellular activity and ECM formation, enabling waste removal, nutrient delivery, and oxygen diffusion. These 3D structures also offer a structural framework to withstand external forces and gradually remodel as new bone tissue forms [ 27 ]. Customized 3D-printed scaffolds are crucial for regenerating CSDs with complex geometries [ 28 ]. Synthetic polymers such as polycaprolactone (PCL), polylactic-glycolic acid (PLGA), and polylactic acid (PLA) are widely used materials for the 3D printing of scaffolds in BTE [ 29 ]. Among them, PCL is well suited for scaffold fabrication due to its thermoelastic behavior, mechanical strength, and controlled structural degradation time proportional to recovery. However, PCL has the disadvantages of low flexibility, lack of cell attachment sites, low performance, and the absence of functional groups on the polymer chain compared to natural bone tissue [ 30 – 32 ]. Hydroxyapatite (HA) is a calcium phosphate with excellent biocompatibility, osteoconductivity, biocompatibility, and cell adhesion and proliferation properties, making it very suitable as a bone substitute [ 31 , 32 ]. Numerous experiments have been conducted on biomedical scaffolds combining PCL and HA for BTE applications [ 33 , 34 ]. Jiao et al. [ 33 ] and Kim et al. [ 34 ] reported that 3D-printed PCL/HA scaffolds increased the mechanical strength of the scaffolds compared with PCL scaffolds alone. Therefore, a combination of PCL and HA can provide adequate mechanical strength to the scaffolds. Nevertheless, these 3D-printed polymer scaffolds typically have low osteoinductive capacity, which results in limited osteogenesis [ 26 , 35 ]. Current research is focused on increasing the osteoinductive potential of this 3D-printed scaffold to promote bone healing. One novel approach currently being investigated is the use of a synergistic strategy of incorporating MSC spheroids into 3D-printed scaffolds to improve the osteoinductive potential of the scaffold. Promoting bone regeneration in complex CSD is critical to effectively treating patients and reducing long-term healthcare costs. Despite the growing interest in the synergistic strategy of MSC spheroids and scaffolds, few studies have investigated this strategy in vivo for bone tissue regeneration [ 36 , 37 ]. Therefore, the main objective of this study is to evaluate the efficacy of ADSC-spheroids within a 3D-printed scaffold made of PCL and HA in repairing CSDs utilizing a rabbit radial defect model. Results Spheroid formation and cell viability. ADSC-spheroids were successfully established in silicone elastomer-based concave microwells after one day of culture (Fig. 1 ). Cell viability was assessed using a fluorescence-based live/dead assay. Viable cells emit green fluorescence, while dead cells exhibit red fluorescence. On day 1, most ADSC-single cells and spheroids displayed green fluorescence, indicating high viability. By days 4 and 7, an increased red fluorescence was observed in both groups as the incubation time increased; there was a propensity for more dead cells among ADSC-single cells than among ADSC-spheroids. Alizarin Red S staining analysis. Alizarin Red S staining was conducted to assess the level of calcification after 7 and 14 days of osteoblast differentiation (Fig. 2 a). The relative values of the Alizarin Red S-stained area at day 7 were 7.03 ± 1.41, 56.95 ± 0.72, and 69.23 ± 1.58% for the No cells, ADSC-single cell, and ADSC-spheroid groups, respectively, and on day 14, they were 6.0 ± 1.51, 86.86 ± 3.10, and 100.37 ± 2.10%, respectively (Fig. 2 b). The Alizarin Red S-stained area in the ADSC-single cell and ADSC-spheroid groups was significantly higher than that in No cells group on days 7 and 14, with a significant increase on day 14 compared with day 7. On days 7 and 14, the ADSC-spheroid group had the most Alizarin Red S staining among the groups. Alkaline phosphatase (ALP) activity analysis. ALP activity was measured after 7 and 14 days of osteoblast differentiation. At day 7, the ALP activity was found to be 33.37 ± 0.25 ng/ml for the ADSC-single cell group and 45.0 ± 0.61 ng/ml for the ADSC-spheroid group. By day 14, the values were 43.07 ± 2.54 ng/ml for the ADSC-single cell group and 70.38 ± 0.48 ng/ml for the ADSC-spheroid group. The ALP activity was significantly increased on day 14 compared with day 7 in both the ADSC-single cell and ADSC-spheroid groups. On day 14, the ADSC-spheroid group showed significantly higher ALP activity than the ADSC-single cell group (Fig. 2 c). Radiographic analysis. Both plain radiographs and micro-computed tomography (micro-CT) images showed that the 3D-printed scaffolds were well-maintained within the defect area until eight weeks post-implantation in all experimental groups. New bone formation started from the margins of the defect (Fig. 3 a). Quantitative analysis using micro-CT showed that the new bone volume (NBV, mm³) of the Control, No cells, ADSC-single cell, and ADSC-spheroid groups were 57.83 ± 8.33 mm³, 83.83 ± 10.23 mm³, 87.35 ± 9.55 mm³, and 110.19 ± 12.13 mm³, respectively, on day 7. On day 14, the NBVs of the Control, No cells, ADSC-single cell, and ADSC-spheroid groups were 94.08 ± 6.65 mm³, 112.84 ± 12.43 mm³, 117.82 ± 11.04 mm³, and 145.58 ± 6.48 mm³, respectively. This micro-CT analysis showed a tendency for more new bone formation in the ADSC-spheroid group than in the No cells and ADSC-single cell groups at weeks 4 and 8; however, this difference was not significant. The ADSC-spheroid group showed significantly more new bone formation than the Control group (Fig. 3 b). Histological analysis. Histological examination indicated no adverse reactions, such as inflammation, at the scaffold implantation site for any experimental group at 4 and 8 weeks post-implantation. At 4 weeks post-implantation, minor bone regeneration was observed at the defect margin in all groups. At 8 weeks post-implantation, bone regeneration was confirmed at the defect margin in the Control group. In the other experimental groups, new bone formation was evident at the margins and center of the defect site (Fig. 4 ). The percentage of NBV was analyzed by quantitatively measuring the stained area of new bone within the defect using an ImageJ-converted image. At week 4, the percentage NBV was calculated to be 4.47 ± 0.63%, 7.55 ± 1.08%, 8.73 ± 1.01%, and 10.8 ± 0.48% for the Control, No cells, ADSC-single cell, and ADSC-spheroid groups, respectively. At week 8, the Control, No cells, ADSC-single cell, and ADSC-spheroid groups showed percentage NBVs of 9.76 ± 1.41%, 13.44 ± 0.89%, 14.62 ± 1.46%, and 16.33 ± 0.85%, respectively. This analysis showed a tendency for more new bone formation in the ADSC-spheroid group than in the No cells and ADSC-single cell groups at weeks 4 and 8; however, this difference was not significant. The ADSC-spheroid group showed significantly more new bone formation than the Control group (Fig. 5 ). Discussion Stem cell-based therapies are emerging as promising solutions to treat bone defects due to the capacity of MSCs to differentiate into multiple cell types including osteoblasts [ 8 ]. MSC culture techniques are crucial for effectively expressing the osteoinductive properties of MSCs. Traditional 2D monolayer culture techniques cannot create a physiologically suitable 3D microenvironment for MSC differentiation [ 16 ]. Three-dimensional spheroid culture techniques attract attention in tissue engineering because they promote cell-to-cell interactions to form spheroids, facilitating excellent cell differentiation [ 18 ]. Recent studies have shown that the expression of cytokines, such as fibroblast growth factor 2, angiogenin, angiopoietin 2, hepatocyte growth factor, and vascular endothelial growth factors, is also significantly increased in MSC spheroids, which provides a 3D microenvironment that is favorable for the differentiation of MSCs in the body [ 38 – 40 ]. A spheroid formed by the aggregation of MSCs can improve the therapeutic potential of MSCs for the regeneration and repair of bone defects. Considering the abovementioned advantages of MSC spheroids, it was expected that superior osteogenic differentiation would be observed in vitro in ADSC-spheroids over ADSC-single cells. ALP activity, a well-studied early marker of osteogenic differentiation, was utilized to study the osteogenic differentiation capacity of spheroids [ 41 , 42 ]. In addition, Alizarin Red S staining was used as an indicator of osteogenic maturation of ADSCs to visualize and quantify the presence of a calcified matrix in the cell [ 43 , 44 ]. This study showed that ADSC-spheroids exhibited relatively more osteogenic activity than ADSC-single cells when measured by ALP activity and Alizarin Red S levels (Fig. 5 ). This result is thought to be related to accelerated osteogenesis and mineralization due to increased cell-cell contact and bone-specific ECM secretion in spheroids [ 45 ]. Overall, the results of this study are in accordance with those of other studies demonstrating osteogenic differentiation of MSC spheroids. For instance, Li et al. [ 46 ] compared the ALP activity between single cells and spheroids of alveolar bone-derived MSCs (AB-MSCs). They reported that the AB-MSC-spheroids had significantly higher ALP activity than the AB-MSC-single cells after 14 days of culture. Shanbhag et al. [ 36 ] compared the level of mineralization of single cells and spheroids of bone marrow MSCs (BMSCs) using an in vitro Alizarin Red S staining assay. They reported that a higher mineralization tendency was observed in BMSC-spheroids than in BMSC-single cells after 21 days of culture. Similar results were also observed in a study using human ADSCs (hADSCs) conducted by Gurumurthy et al. [ 47 ]. However, transplanting stem cell spheroids into bone defects in vivo remains challenging. The most effective spheroid delivery method to the regeneration site has not been thoroughly studied. Traditional in vivo administration of spheroids involves seeding cells directly onto a scaffold before implantation. However, this direct seeding method makes it difficult to evenly distribute cells on the scaffold and maintain their function [ 48 ]. Several recent studies [ 49 – 51 ] have demonstrated superior cell function and osteogenesis in vitro and in vivo by encapsulating MSC spheroids in hydrogels. Unlike direct seeding, encapsulating spheroids in hydrogels maintains cell function during in vivo implantation. In this study, collagen gel was used as a hydrogel carrier to encapsulate the cells and maintain cell function during in vivo implantation. The cell-containing hydrogels were applied to complex bone defects along with rigid polymer scaffolds to ensure stable cell maintenance in vivo . Three-dimensional printing technology offers an excellent opportunity to create customized 3D-printed scaffolds for treating complex bone defects. A primary advantage of 3D printing is the capacity to fabricate scaffolds with complex porous structures. Porous scaffold networks with interconnected structures help cell migration, growth, and promotion [ 52 ]. Increasing pore size enhances osteogenesis by facilitating vascularization; nevertheless, it concurrently diminishes the structural mechanical integrity [ 33 , 53 , 54 ]. Some studies have reported that implants with pore sizes larger than 300 µm have better cell differentiation, proliferation, migration, nutrient delivery, and osteogenesis [ 33 , 53 , 55 ]. Wang et al. applied a scaffold with a pore size of 200–500 µm to femoral shaft defects in dogs and found it to be osteoinductive with good biocompatibility [ 56 ]. A customized 3D-printed scaffold is essential for the attachment and proliferation of anchorage-dependent osteoblasts. If the scaffold and host bone meet tightly without gaps, new bone formation can be promoted outward from the host bone [ 57 ]. In this respect, customized 3D-printed scaffolds with complex geometries are crucial for filling and repairing complex CSDs. To evaluate the potential of bone regeneration in vivo , customized 20-mm long 3D-printed scaffolds were fabricated with a pore size of 500 µm. The efficacy of the scaffolds containing ADSC-single cells or spheroids was assessed using a 20-mm long radial defect model in rabbits. The scaffolds matched the defect area well and were firmly connected to the host bone without gaps. In the radiographic analysis, the 3D-printed scaffolds were securely positioned in the defect, and new bone formation occurred over the scaffolds from the defect margin at weeks 4 and 8 post-implantation. Quantitative analysis using micro-CT showed a non-significant tendency for increased new bone formation in the ADSC-spheroid group compared with the No cells and ADSC-single cell groups at weeks 4 and 8. Only the ADSC-spheroid group showed significantly greater new bone formation than the Control group. Histological analysis did not reveal any specific inflammatory responses in any group, and the results of new bone formation between the groups were similar to the micro-CT analysis results. This high osteogenic tendency of ADSC-spheroids is thought to be due to the superior in situ mineralization of the implanted spheroids. Few studies have explored the synergistic strategy of applying MSC spheroids with 3D-printed scaffolds to bone defects [ 36 , 37 ]. Kronemberger et al. [ 37 ] evaluated the osteogenic effect of the synergistic strategy using 3D-printed scaffolds with manually seeded hADSC-spheroids in a rat calvarial defect model. The researchers found that hADSC-spheroids in 3D-printed scaffolds successfully promoted new bone formation; however, the new bone formation was not significantly greater than that achieved without hADSC-spheroids in the scaffold. Shanbhag et al. [ 36 ] evaluated the osteogenic effect of a synergistic strategy using 3D-printed scaffolds with hydrogels containing either hBMSC-single cells or spheroids in a rat calvarial defect model. Despite a trend for superior in vitro mineralization of hBMSC-spheroids, scaffolds containing hBMSC-single cells or spheroids showed similar osteogenic performance in vivo . In the present study, the 3D-printed scaffolds with collagen hydrogels containing cells were applied to a rabbit radial defect model. The results showed significantly more new bone formation in the ADSC-spheroid group than in the Control group, suggesting that this strategy has the potential for regeneration of complex bone defects. Considering that the ADSC-spheroid group did not show significant differences from the other groups except for the Control group, further studies will need to adjust the number of experimental animals and the delivery method of spheroids in vivo . It should be noted that the scaffold showed no signs of degradation or replacement during the experiment. PCL has been reported to be a promising scaffold material in various tissue engineering applications, but it has a slow degradation rate, taking two to three years to degrade entirely [ 58 ]. Little is known about the in vivo degradation profile of PCL/HA depending on the HA content. The in vivo analysis in this study showed that new bone formation occurred around the scaffold, but the scaffold was not replaced by bone. However, this study did not evaluate scaffold degradation because the experimental period was short. Therefore, further investigations including mechanical tests of scaffold degradation and long-term in vivo evaluation are needed. This is the first study to utilize a rabbit radial defect model based on a synergistic strategy to encourage bone regeneration using ADSC-spheroids combined with 3D-printed PCL/HA scaffolds. The findings of this study revealed that this strategy enhanced the osteogenic differentiation of ADSC-spheroids in vitro and promoted more effective osteogenesis in vivo , showing a significantly greater new bone formation in the ADSC-spheroid group than the Control group. Future research should optimize the delivery methods of ADSC-spheroids to 3D-printed scaffolds, potentially exploring techniques including 3D bioprinting to improve in vivo regenerative responses. In addition, more comprehensive studies, including immunohistochemistry staining for bone regeneration proteins and long-term follow-ups, will be crucial in thoroughly assessing the potential of ADSC-spheroids for in vivo bone regeneration. If future investigations can demonstrate the in vivo effectiveness of ADSC-spheroids while addressing the abovementioned limitations, it could be a significant breakthrough in treating complex bone defects in orthopedics. Methods Fabrication of 3D-printed scaffold. Three-dimensional-printed scaffolds were fabricated using PCL (Polysciences, Warrington, PA, USA) and HA derived from porcine femoral cancellous bone. A PCL/HA blend was prepared by mixing 20% (wt) HA powder with molten PCL. After that, the PCL/HA mixture was put into a steel syringe and extruded using a 3DXPrinter (T&R Biofab Co., Ltd, Siheung, Republic of Korea) through a steel nozzle. For the in vitro experiments, the fabricated scaffolds had the following dimensions: 7.8 mm diameter, 1.2 mm height, 300 µm line height, 500 µm pore size, and 300 µm line width (Fig. 6 ). The PCL/HA scaffolds were fabricated, sanitized for 3 hours with 70% ethanol in a 24-well cell culture plate (Falcon® Cell Culture Plate, #353047, Corning Inc., Corning, NY, USA), and then allowed to dry overnight on a clean bench under ultraviolet (UV) light. For in vivo experiments, scaffolds measuring 4 mm in diameter and 20 mm in length were fabricated using the same method to fit the rabbit radial defect model (Fig. 7 ). Cell isolation and 2D monolayer culture. ADSCs were obtained from three-month-old male New Zealand White (NZW) rabbits (weight: 3.0–3.5 kg, Damool Science, Daejeon, Republic of Korea), following procedures approved by the Institutional Animal Care and Use Committee of Chonnam National University in Korea (Approval No. CNU IACUC-YB-2022-149). The adipose tissue harvested from the interscapular area of the rabbits was extensively washed in phosphate-buffered saline (PBS, #14190-144, Thermo Fisher Scientific Inc., Waltham, MA, USA). The tissue was chopped and digested for 1 hour at 37°C in a shaking water bath using 2 mg/ml collagenase type I (#LS004196, Worthington Biochemical Corporation, Worthington, NJ, USA) in Hank's Balanced Salt Solution (HBSS, #14025-092, Gibco Inc., Grand Island, NY, USA). The digested mixture was filtered through a 100-µm cell strainer (Corning® 100 µm cell strainer, #CLS431752, Corning Inc.) and centrifuged at 1600 rpm for 10 minutes. The pelleted cells were resuspended in HBSS, followed by a second centrifugation. The ADSCs were cultured in Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F-12, #11320033, Gibco Inc.) supplemented with 15% fetal bovine serum (FBS, #16000-044, Gibco Inc.) and 1% antibiotics (penicillin-streptomycin, #15140-122, Gibco Inc.) at 37℃ in a 5% CO 2 incubator. The cells were passaged using 0.25% trypsin with EDTA (#SH30042.02, Thermo Scientific Hyclone, Logan, UT, USA), and the media was changed every two days. 3D spheroid culture. The ADSC-spheroids were formed in silicone elastomer-based concave microwells (StemFIT 3D, #H853400, MicroFIT, Seongnam, Republic of Korea) with a diameter of 400 µm. A total of 1.2 x 10 6 cells were loaded into each well and cultured in alpha modification of minimal essential medium (α-MEM, #A10490-01, Gibco Inc.) supplemented with 15% FBS and 1% antibiotics (penicillin-streptomycin). The formation and morphological changes of the ADSC-spheroids were observed under an inverted phase contrast fluorescence microscope (Leica DM IL LED Fluo, Leica Microsystems GmbH, Wetzlar, Germany). Cell seeding and differentiation on PCL/HA scaffolds. The collagen gel was prepared by dissolving porcine-derived collagen in acetic acid (Duksan Pure Chemical Co., Ansan, Republic of Korea) for 8 hours. A total of 1.2 × 10 6 cells/ml of ADSC-single cells and spheroids with 0.1 ml collagen gel was seeded onto PCL/HA scaffolds. The scaffolds were cultured in DMEM/F-12 supplemented with 50 µg/ml L-ascorbic acid (#A92902, Sigma-Aldrich, St Louis, MO, USA), 5 mM β-glycerophosphate (#G9422, Sigma-Aldrich), 15% FBS, and 1% antibiotics (penicillin-streptomycin) at 37℃. The osteogenic differentiation medium was replaced every two days during the differentiation period. Cell viability test. The viability of ADSC-single cells and spheroids on the scaffolds was analyzed using a Live/Dead assay kit (#L3224, Invitrogen®, Carlsbad, CA, USA) at days 0, 1, 4, and 7. The ADSC-single cells and spheroids were stained in 1 ml of DMEM/F-12 containing 0.5 µl of calcein acetomethyl ester (4 mM; Invitrogen®) and 2 µl of ethidium homodimer-1 (2 mM; Invitrogen®) for 30 minutes at room temperature. After 30 minutes, the samples were examined under an inverted phase contrast fluorescence microscope. Alizarin red S staining assay. After 7 and 14 days of osteoblast differentiation on the 3D-printed scaffolds, the level of calcification was assessed using Alizarin Red S staining. At the end of the differentiation period, the osteogenic medium was removed, and the cells were washed twice with PBS. A 4% paraformaldehyde (PFA) solution was used to fix the differentiated cells for 20 minutes at room temperature. The cells were washed twice with deionized water after removing the PFA solution. The washed cells were stained with Alizarin Red S staining solution (#20003999, Sigma-Aldrich) for 40 minutes at room temperature. To remove nonspecific staining, the Alizarin Red S solution was removed, and the cells were washed three times with deionized water. The relative area of Alizarin Red S staining was measured utilizing ImageJ software (National Institutes of Health, Bethesda, MD, USA). ALP activity assay. After 7 and 14 days of osteoblast differentiation on the 3D-printed scaffolds, ALP activity assays were performed. A commercially available kit (Senso-Lyte® p-nitrophenyl phosphate alkaline phosphatase assay kit, #AS-72146, AnaSpec Inc., Fremont, CA, USA) was used to evaluate ALP activity according to the manufacturer's instructions. A total of 200 µl/well of biological samples containing ALP was added and incubated at 25°C for 60 minutes to detect osteogenic differentiation. Following incubation, 100 µl of stop solution was added to each well to stop the reaction. The absorbance of the resultant p-nitrophenol was measured using spectrophotometry at 405 nm. Experimental animals. This study was approved by the Institutional Animal Care and Use Committee of Chonnam National University (Approval No. CNU IACUC-YB-2022-149) and performed in accordance with the relevant guidelines and regulations. The modified Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines were followed in all study procedures. The study included 20 healthy 3-month-old male NZW rabbits (weight: 3.0–3.5 kg, Damool Science, Daejeon, Republic of Korea). The rabbits were housed in a temperature-controlled air-conditioned room (20 ± 2℃) with a relative humidity of 50 ± 10% and a light-dark cycle of 12 hours. During the entire study period, they were provided with a commercial rabbit diet (Damool Science). The 20 rabbits were divided into two groups: 10 animals to be sacrificed in week 4 and another 10 animals to be sacrificed in week 8. The experiment was performed on both forelimbs of all animals. The 20 forelimbs of the 10 animals assigned to each of weeks 4 and 8 were randomly divided into 4 groups, with 5 forelimbs in each group. The four experimental groups were the Control group (only critical-size defect), the No cells group (3D-printed scaffold), the ADSC-single cell group (3D-printed scaffold with ADSC-single cells), and the ADSC-spheroid group (3D-printed scaffold with ADSC-spheroids). Anesthesia and surgical procedure. General anesthesia was achieved through intramuscular injection of 3 mg/kg of xylazine (Rompun ® , Bayer Korea Co., Seoul, Republic of Korea) and 6 mg/kg of alfaxalone (Alfaxan ® , Jurox, Australia). Inhalation anesthesia was maintained using isoflurane (Ifran Liq. 1–2%, Hana Pharm Co., Seoul, Republic of Korea). During the procedure, 0.9% N/S fluid (2–5 ml/kg/hr; Normal Saline Inj., JW Pharm Co., Gwacheon, Republic of Korea) was administered to facilitate blood circulation and prevent unexpected bleeding. Pain was controlled with subcutaneous injections of 10 mg/kg of tramadol (Tramadol HCl Huons Inj., Huons Co., Seongnam, Republic of Korea) and 3 mg/kg of ketoprofen (Ketopro Inj., Unibiotech, Anyang, Republic of Korea). To prevent infection, 10 mg/kg of enrofloxacin (Baytril ® 50 Inj., Bayer Korea Co.) was injected subcutaneously. After shaving, the surgical site was disinfected using a povidone-iodine solution and 70% ethanol. A longitudinal incision in the skin was made along the radius. By dissecting the surrounding muscles, the radius was exposed. A 20-mm radial defect was created along the marking using ultrasonic piezoelectric bone surgery equipment (Surgystar Plus, DMETEC Co., Bucheon, Republic of Korea). Each scaffold was implanted into the defect according to the experimental groups. The scaffolds were fixed to the remaining radius with 27-G surgical wires (Solco Biomedical Co., Pyeongtaek, Republic of Korea). The dissected muscle was sutured with a 4 − 0 polyglyconate suture (Maxon®, Covidien, Dublin, Ireland), and the incised skin was closed with a 4 − 0 polyglycolic acid suture (SurgiSorb®, Samyang Co., Seongnam, Republic of Korea) (Fig. 8 ). After surgery, 1 mg/kg of ketoprofen was administered subcutaneously for analgesia and as an anti-inflammatory, and 10 mg/kg of enrofloxacin was administered for as an antibiotic for 1 week. The surgical site was disinfected with povidone-iodine once daily to prevent infection, and a neck collar was placed on the rabbit until recovery was confirmed in order to prevent the animal from licking or damaging the surgical site. At 4 and 8 weeks post-implantation, anesthesia was induced as previously described, and a high concentration of inhalational anesthesia (isoflurane) was used for deep anesthesia. Euthanasia was performed via intravenous injection of 150 mg/kg of KCl (potassium chloride 40 injection, Dai Han Pharm Co.), and samples were harvested. Radiographic evaluation. Forelimb radiographs were taken immediately before the rabbits were sacrificed. Micro-CT scans were performed on the samples after euthanasia. Micro-CT analysis employed a radiation level of 130 kVp and 60 µA using a microtomograph (SkyScan 1173, Bruker-CT, Kontich, Belgium). Measurements were collected using SkyScan 1173 control software (version 1.6, Bruker-CT) with a tube current of 60 µA and tube voltage of 130 kVp. A total of 800 high-resolution images were captured with a resolution of 2,240 × 2,240 pixels, a pixel size of 24.96 µm, and a rotation angle of 0.3° for a total of 180°, with an exposure time of 500 ms. Data Viewer (Ver. 1.5.6.2, Bruker-CT) was used to arrange the orientation of the section images, and Nrecon (Ver. 1.7.4.6, Bruker-CT) was used to perform section reconstruction. The NBV in the defect site was analyzed using CT Analyzer (Ver. 1.19.4.0, Bruker-CT). A region of interest (ROI) was established to minimize interference from the host bone. Grayscale values ranging from 68 to 255 denoted mineralized tissue, with values between 68 and 99 signifying newly mineralized tissue within the defects [ 59 ]. The NBV was calculated as the sum of newly formed bone volumes within the defect. Histological evaluation. Calci-ClearTM Rapid (National Diagnostics, Atlanta, GA, USA) was used to decalcify the samples after they had been fixed in 10% buffered formalin for 24 hours. The samples were subsequently dehydrated with a series of alcohol rinses before being embedded in paraplast (Sherwood Medical Industries, Deland, FL, USA). Embedded samples were sectioned to a thickness of 5 µm using a microtome (Cambridge Instruments, Germany). The slides were stained with hematoxylin and eosin (H&E) and masson's trichrome (MT) for microscopic analysis. The area of new bone in the MT-stained images was quantitatively analyzed using ImageJ software. Statistical analysis. The data are presented as the mean ± standard error (SE). To evaluate the NBV on micro-CT and histological images, GraphPad Prism 8.0 software (GraphPad Software Inc., Boston, MA, USA) was used to conduct one-way ANOVA and Tukey's post hoc test, and p < 0.05 was considered statistically significant. Declarations Acknowledgements This study was supported by the National Research Foundation (NRF) grant funded by the Korea government (MSIT) (No. 2020R1C1C1009798 and RS-2024-0045442640982119420101). Author Contributions Y.C., K.J., S.S.K. and S.E.K. conceived of the project and designed the research. Y.C., K.J., S.L., Y.K., S.J., K.M.S., S.S.K. and S.E.K. performed the research. Y.C., K.J., S.L., S.S.K. and S.E.K. analyzed data. Y.C., K.J., S.L., S.S.K. and S.E.K. wrote the manuscript. All authors approved the manuscript. Competing Interests The authors declare no competing interests. Ethics declarations The animal study was approved by the Institutional Animal Care and Use Committee of Chonnam National University in Korea (Approval No. CNU IACUC-YB-2022-149). Data Availability Additional inquiries can be referred to the corresponding authors; the article contains the original contributions made in the study References Zeng, A., Li, H., Liu, J. & Wu, M. The Progress of Decellularized Scaffold in Stomatology. Tissue Engineering and Regenerative Medicine 19, 451–461 (2022). Pereira, H. F., Cengiz, I. F., Silva, F. S., Reis, R. L. & Oliveira, J. M. Scaffolds and coatings for bone regeneration. Journal of Materials Science: Materials in Medicine 31, 27 (2020). Park, J. Y. et al. 3D printing technology to control BMP-2 and VEGF delivery spatially and temporally to promote large-volume bone regeneration. Journal of Materials Chemistry B 3, 5415–5425 (2015). Baldwin, P. et al. Autograft, Allograft, and Bone Graft Substitutes: Clinical Evidence and Indications for Use in the Setting of Orthopaedic Trauma Surgery. Journal of Orthopaedic Trauma 33, 203–213 (2019). Zhang, S. et al. Clinical Reference Strategy for the Selection of Treatment Materials for Maxillofacial Bone Transplantation: A Systematic Review and Network Meta-Analysis. Tissue Engineering and Regenerative Medicine 19, 437–450 (2022). Genova, T. et al. Advances on Bone Substitutes through 3D Bioprinting. International Journal of Molecular Sciences 21, 7012 (2020). Cancedda, R., Giannoni, P. & Mastrogiacomo, M. A tissue engineering approach to bone repair in large animal models and in clinical practice. Biomaterials 28, 4240–4250 (2007). Bhattacharyya, A., Khatun, M. R., Narmatha, S., Nagarajan, R. & Noh, I. Modulation of 3D Bioprintability in Polysaccharide Bioink by Bioglass Nanoparticles and Multiple Metal Ions for Tissue Engineering. Tissue Engineering and Regenerative Medicine 21, 261–275 (2023). Friedenstein, A. J., Chailakhyan, R. K., Latsinik, N. V., Panasyuk, A. F. & Keiliss-Borok, I. V. Stromal cells responsible for transferring the microenvironment of the hemopoietic tissues: cloning in vitro and retransplantation in vivo. Transplantation 17, 331–340 (1974). Campagnoli, C. et al. Identification of mesenchymal stem/progenitor cells in human first-trimester fetal blood, liver, and bone marrow. Blood 98, 2396–2402 (2001). Erices, A., Conget, P. & Minguell, J. J. Mesenchymal progenitor cells in human umbilical cord blood. British Journal of Haematology 109, 235–242 (2000). Zuk, P. A. et al. Human Adipose Tissue Is a Source of Multipotent Stem Cells. Molecular Biology of the Cell 13, 4279–4295 (2002). Yamada, Y. et al. 3D-cultured small size adipose-derived stem cell spheroids promote bone regeneration in the critical-sized bone defect rat model. Biochemical and Biophysical Research Communications 603, 57–62 (2022). Reddi, A. H. & Cunningham, N. S. Recent Progress in Bone Induction by Osteogenin and Bone Morphogenetic Proteins: Challenges for Biomechanical and Tissue Engineering. Journal of Biomechanical Engineering 113, 189–190 (1991). Lee, S., Choi, E., Cha, M.-J. & Hwang, K.-C. Cell Adhesion and Long-Term Survival of Transplanted Mesenchymal Stem Cells: A Prerequisite for Cell Therapy. Oxidative Medicine and Cellular Longevity 2015, 1–9 (2015). Colter, D. C., Class, R., DiGirolamo, C. M. & Prockop, D. J. Rapid expansion of recycling stem cells in cultures of plastic-adherent cells from human bone marrow. Proceedings of the National Academy of Sciences 97, 3213–3218 (2000). Lee, S. I., Ko, Y. & Park, J.-B. Evaluation of the osteogenic differentiation of gingiva-derived stem cells grown on culture plates or in stem cell spheroids: Comparison of two- and three-dimensional cultures. Experimental and Therapeutic Medicine 14, 2434–2438 (2017). Achilli, T. M., Meyer, J. & Morgan, J. R. Advances in the formation, use and understanding of multi-cellular spheroids. Expert Opinion on Biological Therapy 12, 1347–1360 (2012). Lee, D. H. & Bhang, S. H. Development of Hetero-Cell Type Spheroids Via Core–Shell Strategy for Enhanced Wound Healing Effect of Human Adipose-Derived Stem Cells. Tissue Engineering and Regenerative Medicine 20, 581–591 (2023). Murphy, K. C., Fang, S. Y. & Leach, J. K. Human mesenchymal stem cell spheroids in fibrin hydrogels exhibit improved cell survival and potential for bone healing. Cell and Tissue Research 357, 91–99 (2014). Bartosh, T. J. et al. Aggregation of human mesenchymal stromal cells (MSCs) into 3D spheroids enhances their anti-inflammatory properties. Proceedings of the National Academy of Sciences 107, 13724–13729 (2010). Bhang, S. H., Lee, S., Shin, J.-Y., Lee, T.-J. & Kim, B.-S. Transplantation of Cord Blood Mesenchymal Stem Cells as Spheroids Enhances Vascularization. Tissue Engineering Part A 18, 2138–2147 (2012). Roddy, E., DeBaun, M. R., Daoud-Gray, A., Yang, Y. P. & Gardner, M. J. Treatment of critical-sized bone defects: clinical and tissue engineering perspectives. European Journal of Orthopaedic Surgery &, doi:Traumatology 28, 351–362 (2017). Shen, F. H. et al. Implications of adipose-derived stromal cells in a 3D culture system for osteogenic differentiation: an in vitro and in vivo investigation. The Spine Journal 13, 32–43 (2013). Lee, J. et al. Human adipose-derived stem cell spheroids incorporating platelet-derived growth factor (PDGF) and bio-minerals for vascularized bone tissue engineering. Biomaterials 255, 120192 (2020). Lee, J. et al. 3D printed micro-chambers carrying stem cell spheroids and pro-proliferative growth factors for bone tissue regeneration. Biofabrication 13, 015011 (2020). Seol, Y. et al. A new method of fabricating robust freeform 3D ceramic scaffolds for bone tissue regeneration. Biotechnology and Bioengineering 110, 1444–1455 (2013). Yeo, A., Sju, E., Rai, B. & Teoh, S. H. Customizing the degradation and load‐bearing profile of 3D polycaprolactone‐tricalcium phosphate scaffolds under enzymatic and hydrolytic conditions. Journal of Biomedical Materials Research Part B: Applied Biomaterials 87B, 562–569 (2008). Liu, N., Zhang, X., Guo, Q., Wu, T. & Wang, Y. 3D Bioprinted Scaffolds for Tissue Repair and Regeneration. Front. Mater. 9, 925321 (2022). Zhang, W. et al. Fabrication and characterization of porous polycaprolactone scaffold via extrusion-based cryogenic 3D printing for tissue engineering. Materials & Design 180, 107946 (2019). Siddiqui, N., Asawa, S., Birru, B., Baadhe, R. & Rao, S. PCL-Based Composite Scaffold Matrices for Tissue Engineering Applications. Mol Biotechnol 60, 506–532 (2018). Rosales-Ibáñez, R. et al. Assessment of a PCL-3D Printing-Dental Pulp Stem Cells Triplet for Bone Engineering: An In Vitro Study. Polymers 13, 1154 (2021). Jiao, Z. et al. 3D printing of HA / PCL composite tissue engineering scaffolds. Advanced Industrial and Engineering Polymer Research 2, 196–202 (2019). Kim, C. G. et al. Fabrication of Biocompatible Polycaprolactone–Hydroxyapatite Composite Filaments for the FDM 3D Printing of Bone Scaffolds. Applied Sciences 11, 6351 (2021). JEONG, K. J. et al. Successful Clinical Application of Cancellous Allografts With Structural Support for Failed Bone Fracture Healing in Dogs. In Vivo 33, 1813–1818 (2019). Shanbhag, S. et al. Bone regeneration in rat calvarial defects using dissociated or spheroid mesenchymal stromal cells in scaffold-hydrogel constructs. Stem Cell Res Ther 12, 575 (2021). Kronemberger, G. S. et al. A Synergic Strategy: Adipose-Derived Stem Cell Spheroids Seeded on 3D-Printed PLA/CHA Scaffolds Implanted in a Bone Critical-Size Defect Model. JFB 14, 555 (2023). Kusuma, G. D., Carthew, J., Lim, R. & Frith, J. E. Effect of the microenvironment on mesenchymal stem cell paracrine signaling: opportunities to engineer the therapeutic effect. Stem Cells Dev 26, 617-631 (2017) Potapova, I. A. et al . Mesenchymal stem cells support migration, extracellular matrix invasion, proliferation, and survival of endothelial cells in vitro. Stem Cells 25, 1761-1768 (2007) Zhao, N. et al. Exogenous signaling molecules released from aptamer-functionalized hydrogels promote the survival of mesenchymal stem cell spheroids. ACS Appl. Mater. Interfaces 12, 24599-24610 (2020) Jafary, F., Hanachi, P. & Gorjipour, K. Osteoblast Differentiation on Collagen Scaffold with Immobilized Alkaline Phosphatase. Int J Organ Transplant Med 8, 195-202 (2017). Hashemibeni, B. et al. Comparison of Phenotypic Characterization between Differentiated Osteoblasts from Stem Cells and Calvaria Osteoblasts In vitro. Int J Prev Med 4, 180-186 (2013). Gregory, C. A., Grady Gunn, W., Peister, A. & Prockop, D. J. An Alizarin red-based assay of mineralization by adherent cells in culture: comparison with cetylpyridinium chloride extraction. Analytical Biochemistry 329, 77–84 (2004). Kim, Y. B. & Kim, G. H. PCL/Alginate Composite Scaffolds for Hard Tissue Engineering: Fabrication, Characterization, and Cellular Activities. ACS Comb. Sci. 17, 87–99 (2015). Langenbach, F. et al . Scaffold-free microtissues: differences from monolayer cultures and their potential in bone tissue engineering. Clin Oral Investig 17, 9-17 (2013). Li, N. et al. Spontaneous spheroids from alveolar bone-derived mesenchymal stromal cells maintain pluripotency of stem cells by regulating hypoxia-inducible factors. Biol Res 56, 17 (2023). Gurumurthy, B., Bierdeman, P. C. & Janorkar, A. V. Spheroid model for functional osteogenic evaluation of human adipose derived stem cells. J Biomedical Materials Res 105, 1230–1236 (2017). Villalona, G. A. et al. Cell-Seeding Techniques in Vascular Tissue Engineering. Tissue Engineering Part B: Reviews 16, 341–350 (2010). Ho, S. S., Hung, B. P., Heyrani, N., Lee, M. A. & Leach, J. K. Hypoxic Preconditioning of Mesenchymal Stem Cells with Subsequent Spheroid Formation Accelerates Repair of Segmental Bone Defects. Stem Cells 36, 1393–1403 (2018). Ho, S. S., Keown, A. T., Addison, B. & Leach, J. K. Cell Migration and Bone Formation from Mesenchymal Stem Cell Spheroids in Alginate Hydrogels Are Regulated by Adhesive Ligand Density. Biomacromolecules 18, 4331–4340 (2017). Ho, S. S., Murphy, K. C., Binder, B. Y. K., Vissers, C. B. & Leach, J. K. Increased Survival and Function of Mesenchymal Stem Cell Spheroids Entrapped in Instructive Alginate Hydrogels. Stem Cells Translational Medicine 5, 773–781 (2016). Lee, C. F. et al. 3D Printing of Collagen/Oligomeric Proanthocyanidin/Oxidized Hyaluronic Acid Composite Scaffolds for Articular Cartilage Repair. Polymers 13, 3123 (2021). Jayatissa, N. U. & Bhaduri, S. 3D Printed Polymer Scaffolds for Bone Tissue Regeneration. J Emerg Invest 26, 2 (2019). Marouf, N., Nojehdehian, H. & Ghorbani, F. Physicochemical properties of chitosan–hydroxyapatite matrix incorporated with Ginkgo biloba -loaded PLGA microspheres for tissue engineering applications. Polymers and Polymer Composites 28, 320–330 (2019). Li, H., Tan, C. & Li, L. Review of 3D printable hydrogels and constructs. Materials & Design 159, 20–38 (2018). Wang, X. et al. Three-Dimensional, MultiScale, and Interconnected Trabecular Bone Mimic Porous Tantalum Scaffold for Bone Tissue Engineering. ACS Omega 5, 22520–22528 (2020). Ma, P. X. Biomimetic materials for tissue engineering. Advanced Drug Delivery Reviews 60, 184–198, (2008). Deshpande, M. V., Girase, A. & King, M. W. Degradation of Poly(ε-caprolactone) Resorbable Multifilament Yarn under Physiological Conditions. Polymers 15, 3819 (2023). Jo, H. M. et al. Application of modified porcine xenograft by collagen coating in the veterinary field: pre-clinical and clinical evaluations. Front. Vet. Sci. 11, 1373099 (2024). Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 16 Dec, 2025 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 12 May, 2025 Reviews received at journal 09 May, 2025 Reviewers agreed at journal 30 Apr, 2025 Reviews received at journal 17 Apr, 2025 Reviewers agreed at journal 08 Apr, 2025 Reviewers agreed at journal 04 Apr, 2025 Reviewers invited by journal 04 Apr, 2025 Editor assigned by journal 04 Apr, 2025 Editor invited by journal 03 Apr, 2025 Submission checks completed at journal 02 Apr, 2025 First submitted to journal 24 Mar, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6291864","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":444635548,"identity":"6601a4ad-3d88-499e-93e3-47501aeb0e99","order_by":0,"name":"Yangwon Chae","email":"","orcid":"","institution":"Department of Veterinary Surgery, College of Veterinary Medicine and BK21 Plus Project Team, Chonnam National University","correspondingAuthor":false,"prefix":"","firstName":"Yangwon","middleName":"","lastName":"Chae","suffix":""},{"id":444635549,"identity":"1578c79e-a18c-4b2a-9183-5635c12b3bd1","order_by":1,"name":"Kwangsik Jang","email":"","orcid":"","institution":"Department of Veterinary Surgery, College of Veterinary Medicine and BK21 Plus Project Team, Chonnam National University","correspondingAuthor":false,"prefix":"","firstName":"Kwangsik","middleName":"","lastName":"Jang","suffix":""},{"id":444635554,"identity":"48dc1f1e-dd77-4d56-9d90-9aeab40ee9b2","order_by":2,"name":"Sol Lee","email":"","orcid":"","institution":"Biomaterial R\u0026BD Center, Chonnam National University","correspondingAuthor":false,"prefix":"","firstName":"Sol","middleName":"","lastName":"Lee","suffix":""},{"id":444635555,"identity":"4f072e32-a66f-4e32-9fad-5740045848b8","order_by":3,"name":"Yong-hun Kim","email":"","orcid":"","institution":"Research Institute, T\u0026R Biofab Co., Ltd.","correspondingAuthor":false,"prefix":"","firstName":"Yong-hun","middleName":"","lastName":"Kim","suffix":""},{"id":444635557,"identity":"2446f9e8-63c3-4f7b-8585-d6d7024d7860","order_by":4,"name":"Songwan Jin","email":"","orcid":"","institution":"Department of Mechanical Engineering, Tech University of Korea","correspondingAuthor":false,"prefix":"","firstName":"Songwan","middleName":"","lastName":"Jin","suffix":""},{"id":444635559,"identity":"5674e183-10fb-4866-93d7-c6161e3646c1","order_by":5,"name":"Kyung Mi Shim","email":"","orcid":"","institution":"Department of Veterinary Surgery, College of Veterinary Medicine and BK21 Plus Project Team, Chonnam National University","correspondingAuthor":false,"prefix":"","firstName":"Kyung","middleName":"Mi","lastName":"Shim","suffix":""},{"id":444635560,"identity":"a2e467b9-be11-43ee-b6d4-03478d2ab6fd","order_by":6,"name":"Seong Soo Kang","email":"","orcid":"","institution":"Department of Veterinary Surgery, College of Veterinary Medicine and BK21 Plus Project Team, Chonnam National University","correspondingAuthor":false,"prefix":"","firstName":"Seong","middleName":"Soo","lastName":"Kang","suffix":""},{"id":444635561,"identity":"17a08525-e343-4a92-a987-fd7fbab64108","order_by":7,"name":"Se Eun Kim","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAwUlEQVRIie3NsQrCMBCA4SsBXQquCYU+Q6SDFEqf5SCQqeDasZNTwbWPUwjURexacLH4AopLQAdbtHPSTTD/chzcxwG4XD8Y/YyEfHe0JSjnEzXtFoTtVUPvul1uKHo3DXJrJAGVklV4JnGFhJWQxYWJhNSPIn8gvEMIAHJu/BKu2kf0wtNIyNOKBJB5V8B6JIvhS2YmrJLrvpSC8GO/i0suzYS26lLrJBX8IFSnc2EmUwLAKwDsAUA649blcrn+rTezrjT6HsmuzgAAAABJRU5ErkJggg==","orcid":"","institution":"Department of Veterinary Surgery, College of Veterinary Medicine and BK21 Plus Project Team, Chonnam National University","correspondingAuthor":true,"prefix":"","firstName":"Se","middleName":"Eun","lastName":"Kim","suffix":""}],"badges":[],"createdAt":"2025-03-24 05:38:18","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6291864/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6291864/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-025-25581-5","type":"published","date":"2025-12-16T15:57:22+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":81396429,"identity":"15911d70-80b7-4947-a8e1-3c1004231909","added_by":"auto","created_at":"2025-04-25 15:38:35","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1107781,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFormation of spheroids of adipose-derived stem cells in microwells\u003c/strong\u003e\u003cbr\u003e\n \u003cstrong\u003eSpheroids of adipose-derived stem cells \u003c/strong\u003ewere successfully formed in silicone elastomer-based concave microwells after one day of culture.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-6291864/v1/7d8d7ed5a4bfc61e65b1fee7.png"},{"id":81395177,"identity":"e4822ac0-c0de-4888-a8a2-0c78a75f9edd","added_by":"auto","created_at":"2025-04-25 15:30:35","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":487049,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eIn vitro\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e evaluation of 3D-printed scaffolds with single cells and spheroids of adipose-derived stem cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(a) Alizarin red S-stained images showing calcium deposition. (b) Graph of the Alizarin Red S staining assay. (c) Graph of\u003cstrong\u003e alkaline phosphatase (ALP) \u003c/strong\u003eactivity\u003cem\u003e\u003cstrong\u003e \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eassay\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003eADSC: Adipose-derived stem cell; No cells group: 3D-printed scaffold; ADSC-single cell group: 3D-printed scaffold with ADSC-single cells; ADSC-spheroid group: 3D-printed scaffold with ADSC-spheroids. Data are presented as the mean ± SE. Statistical significance is indicated as \u003csup\u003e\u003cstrong\u003e†\u003c/strong\u003e\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, \u003csup\u003e\u003cstrong\u003e††\u003c/strong\u003e\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01 vs. No cells group, ##p \u0026lt; 0.01 vs. ADSC-single cell group, and *p \u0026lt; 0.05, ***p \u0026lt; 0.001 vs. Day 7.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-6291864/v1/7f4201d9d194865a2a8b0762.png"},{"id":81396428,"identity":"849a4193-9933-41a1-a5bb-db4a4d11a6a6","added_by":"auto","created_at":"2025-04-25 15:38:35","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1392546,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRadiographic analysis of rabbit\u003c/strong\u003e \u003cstrong\u003eradial defects\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(a) Plain radiographs, micro-computed tomography (micro-CT) images, and 3D images of the rabbit radial defects eight weeks post-implantation. \u003c/strong\u003e(b)\u003cstrong\u003e \u003c/strong\u003eMicro-CT quantification of new bone volume (mm³) at 4 and 8 weeks post-implantation.\u003c/p\u003e\n\u003cp\u003eADSC: Adipose-derived stem cell; Control group: only critical-size defect; No cells group: 3D-printed scaffold; ADSC-single cell group: 3D-printed scaffold with ADSC-single cells; ADSC-spheroid group: 3D-printed scaffold with ADSC-spheroids. Data are presented as the mean ± SE. Statistical significance is indicated as \u003csup\u003e*\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 vs Control group.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-6291864/v1/6ca3c5eabe5996145f54777a.png"},{"id":81395175,"identity":"45d64d6f-3cef-435a-969f-d24a62d6294a","added_by":"auto","created_at":"2025-04-25 15:30:35","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2577772,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHistological images of rabbit\u003c/strong\u003e \u003cstrong\u003eradial defects\u003c/strong\u003e\u003cbr\u003e\n (a) Hematoxylin and eosin (H\u0026amp;E)-stained images 8 weeks post-implantation. (b) Masson's trichrome (MT)\u003cstrong\u003e-\u003c/strong\u003estained images 8 weeks post-implantation.\u003c/p\u003e\n\u003cp\u003eADSC: Adipose-derived stem cell; \u003cstrong\u003eControl group:\u003c/strong\u003e only critical-size defect; \u003cstrong\u003eNo cells group: \u003c/strong\u003e3D-printed scaffold; \u003cstrong\u003eADSC-single cell group:\u003c/strong\u003e 3D-printed scaffold with ADSC-single cells; \u003cstrong\u003eADSC-spheroid group:\u003c/strong\u003e 3D-printed scaffold with ADSC-spheroids; \u003cstrong\u003eArrows:\u003c/strong\u003edefect margin; \u003cstrong\u003enb:\u003c/strong\u003e new bone; \u003cstrong\u003eob:\u003c/strong\u003e old bone; \u003cstrong\u003ect:\u003c/strong\u003econnective tissue.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-6291864/v1/3b8ca3308d9f98bb5777ff1b.png"},{"id":81396431,"identity":"a8c6810a-f97b-4b8c-808d-17c850076e20","added_by":"auto","created_at":"2025-04-25 15:38:35","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1096328,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHistological analysis results of rabbit\u003c/strong\u003e \u003cstrong\u003eradial defects\u003c/strong\u003e\u003cbr\u003e\n(a) Masson's trichrome (MT)-stained images and ImageJ-converted images of rabbit radial defects at 8 weeks post-implantation. (b) ImageJ analysis quantification of the percentage of new bone volume (%) at 4 and 8 weeks post-implantation.\u003c/p\u003e\n\u003cp\u003eADSC: Adipose-derived stem cell; \u003cstrong\u003eControl group:\u003c/strong\u003e only critical-size defect; \u003cstrong\u003eNo cells group:\u003c/strong\u003e 3D-printed scaffold; \u003cstrong\u003eADSC-single cell group:\u003c/strong\u003e 3D-printed scaffold with ADSC-single cells; \u003cstrong\u003eADSC-spheroid group:\u003c/strong\u003e 3D-printed scaffold with ADSC-spheroids; \u003cstrong\u003eArrows:\u003c/strong\u003e defect margin. Data are presented as the mean ± SE. Statistical significance is indicated as \u003csup\u003e*\u003c/sup\u003e\u003cem\u003ep \u0026lt; 0.05 and\u003c/em\u003e \u003csup\u003e** \u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u003cstrong\u003e \u0026lt; 0.01\u003c/strong\u003e vs Control group.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-6291864/v1/2fd61f14d0f777a564aa24c3.png"},{"id":81395184,"identity":"62a173fc-5b0b-4a11-915c-49ea6f0fd347","added_by":"auto","created_at":"2025-04-25 15:30:35","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1817558,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e3D-printed scaffold fabrication for \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein vitro\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e experiments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(a) The 3DXPrinter used to fabricate the scaffold (T\u0026amp;R Biofab Co., Ltd, Siheung, Republic of Korea). (b, c) Top and orthogonal views of the scaffold designed for \u003cem\u003ein vitro\u003c/em\u003e experiments.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-6291864/v1/31ed71e22d1056bbe805b37a.png"},{"id":81395185,"identity":"93dfc7c9-802e-4315-ab2d-e1763000e61f","added_by":"auto","created_at":"2025-04-25 15:30:35","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":478717,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e3D-printed scaffold fabrication for \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein vivo\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e experiments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(a) Design specifications of the 3D-printed scaffold, featuring a length of 20 mm and a diameter of 4 mm. (b, c) Side and orthogonal views of the scaffolds designed for \u003cem\u003ein vivo\u003c/em\u003e experiments.\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-6291864/v1/24c78d53a868438f535d690a.png"},{"id":81395188,"identity":"c1469323-1f65-4356-ae09-4eb417febe84","added_by":"auto","created_at":"2025-04-25 15:30:35","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":3009351,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eImplantation of the 3D-printed scaffold in a rabbit radial defect model\u003c/strong\u003e\u003cbr\u003e\n (a) Disinfection of the surgical area after shaving. (b) Longitudinal skin incision followed by dissecting the surrounding muscles along the radius. (c, d) Formation of a 20-mm long radial defect. (e) Implantation of each scaffold into the defect according to the experimental groups. (f) Suturing of the muscle and skin.\u003c/p\u003e","description":"","filename":"Figure8.png","url":"https://assets-eu.researchsquare.com/files/rs-6291864/v1/ccfdce720536a4732951b422.png"},{"id":98815247,"identity":"9773eefc-b0dc-4d34-9c88-23d1e294ea82","added_by":"auto","created_at":"2025-12-22 16:14:19","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":16799836,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6291864/v1/27f21bdd-0975-487d-9e17-435de412466a.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Bone regeneration using stem cell spheroids within 3D-printed scaffolds in a rabbit radial defect model","fulltext":[{"header":"Introduction","content":"\u003cp\u003eBone defects resulting from inflammation, trauma, tumors, or congenital anomalies present significant challenges to bone regeneration [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. While bone tissue generally exhibits resilience and self-healing abilities, critical-size defects (CSDs) necessitate therapeutic intervention for proper repair [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Autologous bone grafts are considered the gold standard for treating these defects due to their favorable osteoinductive, osteoconductive, and cellular properties [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. However, this approach has limitations including donor site morbidity, long surgical procedures, and limited availability [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Although alternative bone grafting materials such as allografts, xenografts, and alloplasts can address some of these issues, they have the disadvantage of reduced osteogenic capacity due to their lack of osteoinductive potential. Furthermore, these particle-type bone grafts are less likely to maintain their shape in CSDs, making it difficult to provide a structural framework for new bone formation [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eStem cell-based therapies are emerging as promising solutions in bone tissue engineering (BTE) to enhance osteoinductive potential [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Mesenchymal stem cells (MSCs) are multipotent cells in various connective tissues throughout the body including the fetal liver, umbilical cord blood, adipose tissue, and adult bone marrow [\u003cspan additionalcitationids=\"CR10 CR11\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Adipose-derived stem cells (ADSCs), a type of MSC derived from adipose tissue, can differentiate into osteoblasts and exhibit osteoinductive properties [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Their osteoinductive potential is attributed to their high proliferation rates, low immunogenicity, and capacity to differentiate into multiple cell types [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. While some studies have demonstrated the effectiveness of ADSCs in promoting bone regeneration, ADSC-single cells fabricated via two-dimensional (2D) monolayer cultures have limitations in maintaining cell survival and function over time [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. These limitations may arise from the unphysiological microenvironment created by traditional 2D monolayer culture techniques [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eRecent advances in BTE have revealed the benefits of ADSC-spheroids fabricated via three-dimensional (3D) spheroid culture techniques [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. This approach allows for direct cell-to-cell interactions, facilitating the formation of spheroids [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Unlike 2D monolayer cultures, 3D spheroid cultures better replicate the \u003cem\u003ein vivo\u003c/em\u003e environment, enhancing cellular interactions, extracellular matrix (ECM) production, and overall tissue organization [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Moreover, the unique geometry of 3D spheroid cultures can induce anti-inflammatory, anti-apoptotic, and pro-angiogenic effects [\u003cspan additionalcitationids=\"CR21\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. These characteristics enhance the osteoinductive potential of ADSCs in pathological bone repair, particularly in addressing challenges such as avascularity and hypoxia in large defects [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. These improvements in ADSC-spheroid technology could significantly benefit the treatment of CSDs. Several studies [\u003cspan additionalcitationids=\"CR25\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e] have shown that spheroids of ADSCs effectively promote bone growth.\u003c/p\u003e \u003cp\u003eScaffolds serve as 3D structures that provide a transient environment for cellular activity and ECM formation, enabling waste removal, nutrient delivery, and oxygen diffusion. These 3D structures also offer a structural framework to withstand external forces and gradually remodel as new bone tissue forms [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Customized 3D-printed scaffolds are crucial for regenerating CSDs with complex geometries [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Synthetic polymers such as polycaprolactone (PCL), polylactic-glycolic acid (PLGA), and polylactic acid (PLA) are widely used materials for the 3D printing of scaffolds in BTE [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Among them, PCL is well suited for scaffold fabrication due to its thermoelastic behavior, mechanical strength, and controlled structural degradation time proportional to recovery. However, PCL has the disadvantages of low flexibility, lack of cell attachment sites, low performance, and the absence of functional groups on the polymer chain compared to natural bone tissue [\u003cspan additionalcitationids=\"CR31\" citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eHydroxyapatite (HA) is a calcium phosphate with excellent biocompatibility, osteoconductivity, biocompatibility, and cell adhesion and proliferation properties, making it very suitable as a bone substitute [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Numerous experiments have been conducted on biomedical scaffolds combining PCL and HA for BTE applications [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Jiao et al. [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e] and Kim et al. [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e] reported that 3D-printed PCL/HA scaffolds increased the mechanical strength of the scaffolds compared with PCL scaffolds alone. Therefore, a combination of PCL and HA can provide adequate mechanical strength to the scaffolds. Nevertheless, these 3D-printed polymer scaffolds typically have low osteoinductive capacity, which results in limited osteogenesis [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Current research is focused on increasing the osteoinductive potential of this 3D-printed scaffold to promote bone healing. One novel approach currently being investigated is the use of a synergistic strategy of incorporating MSC spheroids into 3D-printed scaffolds to improve the osteoinductive potential of the scaffold.\u003c/p\u003e \u003cp\u003ePromoting bone regeneration in complex CSD is critical to effectively treating patients and reducing long-term healthcare costs. Despite the growing interest in the synergistic strategy of MSC spheroids and scaffolds, few studies have investigated this strategy \u003cem\u003ein vivo\u003c/em\u003e for bone tissue regeneration [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Therefore, the main objective of this study is to evaluate the efficacy of ADSC-spheroids within a 3D-printed scaffold made of PCL and HA in repairing CSDs utilizing a rabbit radial defect model.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eSpheroid formation and cell viability.\u003c/b\u003e ADSC-spheroids were successfully established in silicone elastomer-based concave microwells after one day of culture (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Cell viability was assessed using a fluorescence-based live/dead assay. Viable cells emit green fluorescence, while dead cells exhibit red fluorescence. On day 1, most ADSC-single cells and spheroids displayed green fluorescence, indicating high viability. By days 4 and 7, an increased red fluorescence was observed in both groups as the incubation time increased; there was a propensity for more dead cells among ADSC-single cells than among ADSC-spheroids.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eAlizarin Red S staining analysis.\u003c/b\u003e Alizarin Red S staining was conducted to assess the level of calcification after 7 and 14 days of osteoblast differentiation (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). The relative values of the Alizarin Red S-stained area at day 7 were 7.03\u0026thinsp;\u0026plusmn;\u0026thinsp;1.41, 56.95\u0026thinsp;\u0026plusmn;\u0026thinsp;0.72, and 69.23\u0026thinsp;\u0026plusmn;\u0026thinsp;1.58% for the No cells, ADSC-single cell, and ADSC-spheroid groups, respectively, and on day 14, they were 6.0\u0026thinsp;\u0026plusmn;\u0026thinsp;1.51, 86.86\u0026thinsp;\u0026plusmn;\u0026thinsp;3.10, and 100.37\u0026thinsp;\u0026plusmn;\u0026thinsp;2.10%, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). The Alizarin Red S-stained area in the ADSC-single cell and ADSC-spheroid groups was significantly higher than that in No cells group on days 7 and 14, with a significant increase on day 14 compared with day 7. On days 7 and 14, the ADSC-spheroid group had the most Alizarin Red S staining among the groups.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eAlkaline phosphatase (ALP) activity analysis.\u003c/b\u003e ALP activity was measured after 7 and 14 days of osteoblast differentiation. At day 7, the ALP activity was found to be 33.37\u0026thinsp;\u0026plusmn;\u0026thinsp;0.25 ng/ml for the ADSC-single cell group and 45.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.61 ng/ml for the ADSC-spheroid group. By day 14, the values were 43.07\u0026thinsp;\u0026plusmn;\u0026thinsp;2.54 ng/ml for the ADSC-single cell group and 70.38\u0026thinsp;\u0026plusmn;\u0026thinsp;0.48 ng/ml for the ADSC-spheroid group. The ALP activity was significantly increased on day 14 compared with day 7 in both the ADSC-single cell and ADSC-spheroid groups. On day 14, the ADSC-spheroid group showed significantly higher ALP activity than the ADSC-single cell group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec).\u003c/p\u003e \u003cp\u003e \u003cb\u003eRadiographic analysis.\u003c/b\u003e Both plain radiographs and micro-computed tomography (micro-CT) images showed that the 3D-printed scaffolds were well-maintained within the defect area until eight weeks post-implantation in all experimental groups. New bone formation started from the margins of the defect (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). Quantitative analysis using micro-CT showed that the new bone volume (NBV, mm\u0026sup3;) of the Control, No cells, ADSC-single cell, and ADSC-spheroid groups were 57.83\u0026thinsp;\u0026plusmn;\u0026thinsp;8.33 mm\u0026sup3;, 83.83\u0026thinsp;\u0026plusmn;\u0026thinsp;10.23 mm\u0026sup3;, 87.35\u0026thinsp;\u0026plusmn;\u0026thinsp;9.55 mm\u0026sup3;, and 110.19\u0026thinsp;\u0026plusmn;\u0026thinsp;12.13 mm\u0026sup3;, respectively, on day 7. On day 14, the NBVs of the Control, No cells, ADSC-single cell, and ADSC-spheroid groups were 94.08\u0026thinsp;\u0026plusmn;\u0026thinsp;6.65 mm\u0026sup3;, 112.84\u0026thinsp;\u0026plusmn;\u0026thinsp;12.43 mm\u0026sup3;, 117.82\u0026thinsp;\u0026plusmn;\u0026thinsp;11.04 mm\u0026sup3;, and 145.58\u0026thinsp;\u0026plusmn;\u0026thinsp;6.48 mm\u0026sup3;, respectively. This micro-CT analysis showed a tendency for more new bone formation in the ADSC-spheroid group than in the No cells and ADSC-single cell groups at weeks 4 and 8; however, this difference was not significant. The ADSC-spheroid group showed significantly more new bone formation than the Control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eHistological analysis.\u003c/b\u003e Histological examination indicated no adverse reactions, such as inflammation, at the scaffold implantation site for any experimental group at 4 and 8 weeks post-implantation. At 4 weeks post-implantation, minor bone regeneration was observed at the defect margin in all groups. At 8 weeks post-implantation, bone regeneration was confirmed at the defect margin in the Control group. In the other experimental groups, new bone formation was evident at the margins and center of the defect site (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). The percentage of NBV was analyzed by quantitatively measuring the stained area of new bone within the defect using an ImageJ-converted image. At week 4, the percentage NBV was calculated to be 4.47\u0026thinsp;\u0026plusmn;\u0026thinsp;0.63%, 7.55\u0026thinsp;\u0026plusmn;\u0026thinsp;1.08%, 8.73\u0026thinsp;\u0026plusmn;\u0026thinsp;1.01%, and 10.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.48% for the Control, No cells, ADSC-single cell, and ADSC-spheroid groups, respectively. At week 8, the Control, No cells, ADSC-single cell, and ADSC-spheroid groups showed percentage NBVs of 9.76\u0026thinsp;\u0026plusmn;\u0026thinsp;1.41%, 13.44\u0026thinsp;\u0026plusmn;\u0026thinsp;0.89%, 14.62\u0026thinsp;\u0026plusmn;\u0026thinsp;1.46%, and 16.33\u0026thinsp;\u0026plusmn;\u0026thinsp;0.85%, respectively. This analysis showed a tendency for more new bone formation in the ADSC-spheroid group than in the No cells and ADSC-single cell groups at weeks 4 and 8; however, this difference was not significant. The ADSC-spheroid group showed significantly more new bone formation than the Control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eStem cell-based therapies are emerging as promising solutions to treat bone defects due to the capacity of MSCs to differentiate into multiple cell types including osteoblasts [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. MSC culture techniques are crucial for effectively expressing the osteoinductive properties of MSCs. Traditional 2D monolayer culture techniques cannot create a physiologically suitable 3D microenvironment for MSC differentiation [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Three-dimensional spheroid culture techniques attract attention in tissue engineering because they promote cell-to-cell interactions to form spheroids, facilitating excellent cell differentiation [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Recent studies have shown that the expression of cytokines, such as fibroblast growth factor 2, angiogenin, angiopoietin 2, hepatocyte growth factor, and vascular endothelial growth factors, is also significantly increased in MSC spheroids, which provides a 3D microenvironment that is favorable for the differentiation of MSCs in the body [\u003cspan additionalcitationids=\"CR39\" citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. A spheroid formed by the aggregation of MSCs can improve the therapeutic potential of MSCs for the regeneration and repair of bone defects.\u003c/p\u003e \u003cp\u003eConsidering the abovementioned advantages of MSC spheroids, it was expected that superior osteogenic differentiation would be observed \u003cem\u003ein vitro\u003c/em\u003e in ADSC-spheroids over ADSC-single cells. ALP activity, a well-studied early marker of osteogenic differentiation, was utilized to study the osteogenic differentiation capacity of spheroids [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. In addition, Alizarin Red S staining was used as an indicator of osteogenic maturation of ADSCs to visualize and quantify the presence of a calcified matrix in the cell [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. This study showed that ADSC-spheroids exhibited relatively more osteogenic activity than ADSC-single cells when measured by ALP activity and Alizarin Red S levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). This result is thought to be related to accelerated osteogenesis and mineralization due to increased cell-cell contact and bone-specific ECM secretion in spheroids [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. Overall, the results of this study are in accordance with those of other studies demonstrating osteogenic differentiation of MSC spheroids. For instance, Li et al. [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e] compared the ALP activity between single cells and spheroids of alveolar bone-derived MSCs (AB-MSCs). They reported that the AB-MSC-spheroids had significantly higher ALP activity than the AB-MSC-single cells after 14 days of culture. Shanbhag et al. [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e] compared the level of mineralization of single cells and spheroids of bone marrow MSCs (BMSCs) using an \u003cem\u003ein vitro\u003c/em\u003e Alizarin Red S staining assay. They reported that a higher mineralization tendency was observed in BMSC-spheroids than in BMSC-single cells after 21 days of culture. Similar results were also observed in a study using human ADSCs (hADSCs) conducted by Gurumurthy et al. [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eHowever, transplanting stem cell spheroids into bone defects \u003cem\u003ein vivo\u003c/em\u003e remains challenging. The most effective spheroid delivery method to the regeneration site has not been thoroughly studied. Traditional \u003cem\u003ein vivo\u003c/em\u003e administration of spheroids involves seeding cells directly onto a scaffold before implantation. However, this direct seeding method makes it difficult to evenly distribute cells on the scaffold and maintain their function [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. Several recent studies [\u003cspan additionalcitationids=\"CR50\" citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e] have demonstrated superior cell function and osteogenesis \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e by encapsulating MSC spheroids in hydrogels. Unlike direct seeding, encapsulating spheroids in hydrogels maintains cell function during \u003cem\u003ein vivo\u003c/em\u003e implantation. In this study, collagen gel was used as a hydrogel carrier to encapsulate the cells and maintain cell function during \u003cem\u003ein vivo\u003c/em\u003e implantation. The cell-containing hydrogels were applied to complex bone defects along with rigid polymer scaffolds to ensure stable cell maintenance \u003cem\u003ein vivo\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eThree-dimensional printing technology offers an excellent opportunity to create customized 3D-printed scaffolds for treating complex bone defects. A primary advantage of 3D printing is the capacity to fabricate scaffolds with complex porous structures. Porous scaffold networks with interconnected structures help cell migration, growth, and promotion [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. Increasing pore size enhances osteogenesis by facilitating vascularization; nevertheless, it concurrently diminishes the structural mechanical integrity [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e, \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. Some studies have reported that implants with pore sizes larger than 300 \u0026micro;m have better cell differentiation, proliferation, migration, nutrient delivery, and osteogenesis [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e, \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. Wang et al. applied a scaffold with a pore size of 200\u0026ndash;500 \u0026micro;m to femoral shaft defects in dogs and found it to be osteoinductive with good biocompatibility [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. A customized 3D-printed scaffold is essential for the attachment and proliferation of anchorage-dependent osteoblasts. If the scaffold and host bone meet tightly without gaps, new bone formation can be promoted outward from the host bone [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. In this respect, customized 3D-printed scaffolds with complex geometries are crucial for filling and repairing complex CSDs. To evaluate the potential of bone regeneration \u003cem\u003ein vivo\u003c/em\u003e, customized 20-mm long 3D-printed scaffolds were fabricated with a pore size of 500 \u0026micro;m.\u003c/p\u003e \u003cp\u003eThe efficacy of the scaffolds containing ADSC-single cells or spheroids was assessed using a 20-mm long radial defect model in rabbits. The scaffolds matched the defect area well and were firmly connected to the host bone without gaps. In the radiographic analysis, the 3D-printed scaffolds were securely positioned in the defect, and new bone formation occurred over the scaffolds from the defect margin at weeks 4 and 8 post-implantation. Quantitative analysis using micro-CT showed a non-significant tendency for increased new bone formation in the ADSC-spheroid group compared with the No cells and ADSC-single cell groups at weeks 4 and 8. Only the ADSC-spheroid group showed significantly greater new bone formation than the Control group. Histological analysis did not reveal any specific inflammatory responses in any group, and the results of new bone formation between the groups were similar to the micro-CT analysis results. This high osteogenic tendency of ADSC-spheroids is thought to be due to the superior \u003cem\u003ein situ\u003c/em\u003e mineralization of the implanted spheroids.\u003c/p\u003e \u003cp\u003eFew studies have explored the synergistic strategy of applying MSC spheroids with 3D-printed scaffolds to bone defects [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Kronemberger et al. [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e] evaluated the osteogenic effect of the synergistic strategy using 3D-printed scaffolds with manually seeded hADSC-spheroids in a rat calvarial defect model. The researchers found that hADSC-spheroids in 3D-printed scaffolds successfully promoted new bone formation; however, the new bone formation was not significantly greater than that achieved without hADSC-spheroids in the scaffold. Shanbhag et al. [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e] evaluated the osteogenic effect of a synergistic strategy using 3D-printed scaffolds with hydrogels containing either hBMSC-single cells or spheroids in a rat calvarial defect model. Despite a trend for superior \u003cem\u003ein vitro\u003c/em\u003e mineralization of hBMSC-spheroids, scaffolds containing hBMSC-single cells or spheroids showed similar osteogenic performance \u003cem\u003ein vivo\u003c/em\u003e. In the present study, the 3D-printed scaffolds with collagen hydrogels containing cells were applied to a rabbit radial defect model. The results showed significantly more new bone formation in the ADSC-spheroid group than in the Control group, suggesting that this strategy has the potential for regeneration of complex bone defects. Considering that the ADSC-spheroid group did not show significant differences from the other groups except for the Control group, further studies will need to adjust the number of experimental animals and the delivery method of spheroids \u003cem\u003ein vivo\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eIt should be noted that the scaffold showed no signs of degradation or replacement during the experiment. PCL has been reported to be a promising scaffold material in various tissue engineering applications, but it has a slow degradation rate, taking two to three years to degrade entirely [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e]. Little is known about the \u003cem\u003ein vivo\u003c/em\u003e degradation profile of PCL/HA depending on the HA content. The \u003cem\u003ein vivo\u003c/em\u003e analysis in this study showed that new bone formation occurred around the scaffold, but the scaffold was not replaced by bone. However, this study did not evaluate scaffold degradation because the experimental period was short. Therefore, further investigations including mechanical tests of scaffold degradation and long-term \u003cem\u003ein vivo\u003c/em\u003e evaluation are needed.\u003c/p\u003e \u003cp\u003eThis is the first study to utilize a rabbit radial defect model based on a synergistic strategy to encourage bone regeneration using ADSC-spheroids combined with 3D-printed PCL/HA scaffolds. The findings of this study revealed that this strategy enhanced the osteogenic differentiation of ADSC-spheroids \u003cem\u003ein vitro\u003c/em\u003e and promoted more effective osteogenesis \u003cem\u003ein vivo\u003c/em\u003e, showing a significantly greater new bone formation in the ADSC-spheroid group than the Control group. Future research should optimize the delivery methods of ADSC-spheroids to 3D-printed scaffolds, potentially exploring techniques including 3D bioprinting to improve \u003cem\u003ein vivo\u003c/em\u003e regenerative responses. In addition, more comprehensive studies, including immunohistochemistry staining for bone regeneration proteins and long-term follow-ups, will be crucial in thoroughly assessing the potential of ADSC-spheroids for \u003cem\u003ein vivo\u003c/em\u003e bone regeneration. If future investigations can demonstrate the \u003cem\u003ein vivo\u003c/em\u003e effectiveness of ADSC-spheroids while addressing the abovementioned limitations, it could be a significant breakthrough in treating complex bone defects in orthopedics.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e \u003cb\u003eFabrication of 3D-printed scaffold.\u003c/b\u003e Three-dimensional-printed scaffolds were fabricated using PCL (Polysciences, Warrington, PA, USA) and HA derived from porcine femoral cancellous bone. A PCL/HA blend was prepared by mixing 20% (wt) HA powder with molten PCL. After that, the PCL/HA mixture was put into a steel syringe and extruded using a 3DXPrinter (T\u0026amp;R Biofab Co., Ltd, Siheung, Republic of Korea) through a steel nozzle. For the \u003cem\u003ein vitro\u003c/em\u003e experiments, the fabricated scaffolds had the following dimensions: 7.8 mm diameter, 1.2 mm height, 300 \u0026micro;m line height, 500 \u0026micro;m pore size, and 300 \u0026micro;m line width (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). The PCL/HA scaffolds were fabricated, sanitized for 3 hours with 70% ethanol in a 24-well cell culture plate (Falcon\u0026reg; Cell Culture Plate, #353047, Corning Inc., Corning, NY, USA), and then allowed to dry overnight on a clean bench under ultraviolet (UV) light. For \u003cem\u003ein vivo\u003c/em\u003e experiments, scaffolds measuring 4 mm in diameter and 20 mm in length were fabricated using the same method to fit the rabbit radial defect model (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e\u003cb\u003eCell isolation and 2D monolayer culture.\u003c/b\u003e ADSCs were obtained from three-month-old male New Zealand White (NZW) rabbits (weight: 3.0\u0026ndash;3.5 kg, Damool Science, Daejeon, Republic of Korea), following procedures approved by the Institutional Animal Care and Use Committee of Chonnam National University in Korea (Approval No. CNU IACUC-YB-2022-149). The adipose tissue harvested from the interscapular area of the rabbits was extensively washed in phosphate-buffered saline (PBS, #14190-144, Thermo Fisher Scientific Inc., Waltham, MA, USA). The tissue was chopped and digested for 1 hour at 37\u0026deg;C in a shaking water bath using 2 mg/ml collagenase type I (#LS004196, Worthington Biochemical Corporation, Worthington, NJ, USA) in Hank's Balanced Salt Solution (HBSS, #14025-092, Gibco Inc., Grand Island, NY, USA). The digested mixture was filtered through a 100-\u0026micro;m cell strainer (Corning\u0026reg; 100 \u0026micro;m cell strainer, #CLS431752, Corning Inc.) and centrifuged at 1600 rpm for 10 minutes. The pelleted cells were resuspended in HBSS, followed by a second centrifugation. The ADSCs were cultured in Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F-12, #11320033, Gibco Inc.) supplemented with 15% fetal bovine serum (FBS, #16000-044, Gibco Inc.) and 1% antibiotics (penicillin-streptomycin, #15140-122, Gibco Inc.) at 37℃ in a 5% CO\u003csub\u003e2\u003c/sub\u003e incubator. The cells were passaged using 0.25% trypsin with EDTA (#SH30042.02, Thermo Scientific Hyclone, Logan, UT, USA), and the media was changed every two days.\u003c/p\u003e \u003cp\u003e \u003cb\u003e3D spheroid culture.\u003c/b\u003e The ADSC-spheroids were formed in silicone elastomer-based concave microwells (StemFIT 3D, #H853400, MicroFIT, Seongnam, Republic of Korea) with a diameter of 400 \u0026micro;m. A total of 1.2 x 10\u003csup\u003e6\u003c/sup\u003e cells were loaded into each well and cultured in alpha modification of minimal essential medium (α-MEM, #A10490-01, Gibco Inc.) supplemented with 15% FBS and 1% antibiotics (penicillin-streptomycin). The formation and morphological changes of the ADSC-spheroids were observed under an inverted phase contrast fluorescence microscope (Leica DM IL LED Fluo, Leica Microsystems GmbH, Wetzlar, Germany).\u003c/p\u003e \u003cp\u003e \u003cb\u003eCell seeding and differentiation on PCL/HA scaffolds.\u003c/b\u003e The collagen gel was prepared by dissolving porcine-derived collagen in acetic acid (Duksan Pure Chemical Co., Ansan, Republic of Korea) for 8 hours. A total of 1.2 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e cells/ml of ADSC-single cells and spheroids with 0.1 ml collagen gel was seeded onto PCL/HA scaffolds. The scaffolds were cultured in DMEM/F-12 supplemented with 50 \u0026micro;g/ml L-ascorbic acid (#A92902, Sigma-Aldrich, St Louis, MO, USA), 5 mM β-glycerophosphate (#G9422, Sigma-Aldrich), 15% FBS, and 1% antibiotics (penicillin-streptomycin) at 37℃. The osteogenic differentiation medium was replaced every two days during the differentiation period.\u003c/p\u003e \u003cp\u003e \u003cb\u003eCell viability test.\u003c/b\u003e The viability of ADSC-single cells and spheroids on the scaffolds was analyzed using a Live/Dead assay kit (#L3224, Invitrogen\u0026reg;, Carlsbad, CA, USA) at days 0, 1, 4, and 7. The ADSC-single cells and spheroids were stained in 1 ml of DMEM/F-12 containing 0.5 \u0026micro;l of calcein acetomethyl ester (4 mM; Invitrogen\u0026reg;) and 2 \u0026micro;l of ethidium homodimer-1 (2 mM; Invitrogen\u0026reg;) for 30 minutes at room temperature. After 30 minutes, the samples were examined under an inverted phase contrast fluorescence microscope.\u003c/p\u003e \u003cp\u003e \u003cb\u003eAlizarin red S staining assay.\u003c/b\u003e After 7 and 14 days of osteoblast differentiation on the 3D-printed scaffolds, the level of calcification was assessed using Alizarin Red S staining. At the end of the differentiation period, the osteogenic medium was removed, and the cells were washed twice with PBS. A 4% paraformaldehyde (PFA) solution was used to fix the differentiated cells for 20 minutes at room temperature. The cells were washed twice with deionized water after removing the PFA solution. The washed cells were stained with Alizarin Red S staining solution (#20003999, Sigma-Aldrich) for 40 minutes at room temperature. To remove nonspecific staining, the Alizarin Red S solution was removed, and the cells were washed three times with deionized water. The relative area of Alizarin Red S staining was measured utilizing ImageJ software (National Institutes of Health, Bethesda, MD, USA).\u003c/p\u003e \u003cp\u003e \u003cb\u003eALP activity assay.\u003c/b\u003e After 7 and 14 days of osteoblast differentiation on the 3D-printed scaffolds, ALP activity assays were performed. A commercially available kit (Senso-Lyte\u0026reg; p-nitrophenyl phosphate alkaline phosphatase assay kit, #AS-72146, AnaSpec Inc., Fremont, CA, USA) was used to evaluate ALP activity according to the manufacturer's instructions. A total of 200 \u0026micro;l/well of biological samples containing ALP was added and incubated at 25\u0026deg;C for 60 minutes to detect osteogenic differentiation. Following incubation, 100 \u0026micro;l of stop solution was added to each well to stop the reaction. The absorbance of the resultant p-nitrophenol was measured using spectrophotometry at 405 nm.\u003c/p\u003e \u003cp\u003e\u003cb\u003eExperimental animals.\u003c/b\u003e This study was approved by the Institutional Animal Care and Use Committee of Chonnam National University (Approval No. CNU IACUC-YB-2022-149) and performed in accordance with the relevant guidelines and regulations. The modified Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines were followed in all study procedures. The study included 20 healthy 3-month-old male NZW rabbits (weight: 3.0\u0026ndash;3.5 kg, Damool Science, Daejeon, Republic of Korea). The rabbits were housed in a temperature-controlled air-conditioned room (20\u0026thinsp;\u0026plusmn;\u0026thinsp;2℃) with a relative humidity of 50\u0026thinsp;\u0026plusmn;\u0026thinsp;10% and a light-dark cycle of 12 hours. During the entire study period, they were provided with a commercial rabbit diet (Damool Science). The 20 rabbits were divided into two groups: 10 animals to be sacrificed in week 4 and another 10 animals to be sacrificed in week 8. The experiment was performed on both forelimbs of all animals. The 20 forelimbs of the 10 animals assigned to each of weeks 4 and 8 were randomly divided into 4 groups, with 5 forelimbs in each group. The four experimental groups were the Control group (only critical-size defect), the No cells group (3D-printed scaffold), the ADSC-single cell group (3D-printed scaffold with ADSC-single cells), and the ADSC-spheroid group (3D-printed scaffold with ADSC-spheroids).\u003c/p\u003e \u003cp\u003e \u003cb\u003eAnesthesia and surgical procedure.\u003c/b\u003e General anesthesia was achieved through intramuscular injection of 3 mg/kg of xylazine (Rompun\u003cb\u003e\u0026reg;\u003c/b\u003e, Bayer Korea Co., Seoul, Republic of Korea) and 6 mg/kg of alfaxalone (Alfaxan\u003cb\u003e\u0026reg;\u003c/b\u003e, Jurox, Australia). Inhalation anesthesia was maintained using isoflurane (Ifran Liq. 1\u0026ndash;2%, Hana Pharm Co., Seoul, Republic of Korea). During the procedure, 0.9% N/S fluid (2\u0026ndash;5 ml/kg/hr; Normal Saline Inj., JW Pharm Co., Gwacheon, Republic of Korea) was administered to facilitate blood circulation and prevent unexpected bleeding. Pain was controlled with subcutaneous injections of 10 mg/kg of tramadol (Tramadol HCl Huons Inj., Huons Co., Seongnam, Republic of Korea) and 3 mg/kg of ketoprofen (Ketopro Inj., Unibiotech, Anyang, Republic of Korea). To prevent infection, 10 mg/kg of enrofloxacin (Baytril\u003cb\u003e\u0026reg;\u003c/b\u003e 50 Inj., Bayer Korea Co.) was injected subcutaneously.\u003c/p\u003e \u003cp\u003eAfter shaving, the surgical site was disinfected using a povidone-iodine solution and 70% ethanol. A longitudinal incision in the skin was made along the radius. By dissecting the surrounding muscles, the radius was exposed. A 20-mm radial defect was created along the marking using ultrasonic piezoelectric bone surgery equipment (Surgystar Plus, DMETEC Co., Bucheon, Republic of Korea). Each scaffold was implanted into the defect according to the experimental groups. The scaffolds were fixed to the remaining radius with 27-G surgical wires (Solco Biomedical Co., Pyeongtaek, Republic of Korea). The dissected muscle was sutured with a 4\u0026thinsp;\u0026minus;\u0026thinsp;0 polyglyconate suture (Maxon\u0026reg;, Covidien, Dublin, Ireland), and the incised skin was closed with a 4\u0026thinsp;\u0026minus;\u0026thinsp;0 polyglycolic acid suture (SurgiSorb\u0026reg;, Samyang Co., Seongnam, Republic of Korea) (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAfter surgery, 1 mg/kg of ketoprofen was administered subcutaneously for analgesia and as an anti-inflammatory, and 10 mg/kg of enrofloxacin was administered for as an antibiotic for 1 week. The surgical site was disinfected with povidone-iodine once daily to prevent infection, and a neck collar was placed on the rabbit until recovery was confirmed in order to prevent the animal from licking or damaging the surgical site. At 4 and 8 weeks post-implantation, anesthesia was induced as previously described, and a high concentration of inhalational anesthesia (isoflurane) was used for deep anesthesia. Euthanasia was performed via intravenous injection of 150 mg/kg of KCl (potassium chloride 40 injection, Dai Han Pharm Co.), and samples were harvested.\u003c/p\u003e \u003cp\u003e \u003cb\u003eRadiographic evaluation.\u003c/b\u003e Forelimb radiographs were taken immediately before the rabbits were sacrificed. Micro-CT scans were performed on the samples after euthanasia. Micro-CT analysis employed a radiation level of 130 kVp and 60 \u0026micro;A using a microtomograph (SkyScan 1173, Bruker-CT, Kontich, Belgium). Measurements were collected using SkyScan 1173 control software (version 1.6, Bruker-CT) with a tube current of 60 \u0026micro;A and tube voltage of 130 kVp. A total of 800 high-resolution images were captured with a resolution of 2,240 \u0026times; 2,240 pixels, a pixel size of 24.96 \u0026micro;m, and a rotation angle of 0.3\u0026deg; for a total of 180\u0026deg;, with an exposure time of 500 ms. Data Viewer (Ver. 1.5.6.2, Bruker-CT) was used to arrange the orientation of the section images, and Nrecon (Ver. 1.7.4.6, Bruker-CT) was used to perform section reconstruction. The NBV in the defect site was analyzed using CT Analyzer (Ver. 1.19.4.0, Bruker-CT). A region of interest (ROI) was established to minimize interference from the host bone. Grayscale values ranging from 68 to 255 denoted mineralized tissue, with values between 68 and 99 signifying newly mineralized tissue within the defects [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e]. The NBV was calculated as the sum of newly formed bone volumes within the defect.\u003c/p\u003e \u003cp\u003e \u003cb\u003eHistological evaluation.\u003c/b\u003e Calci-ClearTM Rapid (National Diagnostics, Atlanta, GA, USA) was used to decalcify the samples after they had been fixed in 10% buffered formalin for 24 hours. The samples were subsequently dehydrated with a series of alcohol rinses before being embedded in paraplast (Sherwood Medical Industries, Deland, FL, USA). Embedded samples were sectioned to a thickness of 5 \u0026micro;m using a microtome (Cambridge Instruments, Germany). The slides were stained with hematoxylin and eosin (H\u0026amp;E) and masson's trichrome (MT) for microscopic analysis. The area of new bone in the MT-stained images was quantitatively analyzed using ImageJ software.\u003c/p\u003e \u003cp\u003e \u003cb\u003eStatistical analysis.\u003c/b\u003e The data are presented as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error (SE). To evaluate the NBV on micro-CT and histological images, GraphPad Prism 8.0 software (GraphPad Software Inc., Boston, MA, USA) was used to conduct one-way ANOVA and Tukey's post hoc test, and \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by the National Research Foundation (NRF) grant funded by the Korea government (MSIT) (No. 2020R1C1C1009798 and RS-2024-0045442640982119420101).\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eY.C., K.J., S.S.K. and S.E.K. conceived of the project and designed the research. Y.C., K.J., S.L., Y.K., S.J., K.M.S., S.S.K. and S.E.K. performed the research. Y.C., K.J., S.L., S.S.K. and S.E.K. analyzed data. Y.C., K.J., S.L., S.S.K. and S.E.K. wrote the manuscript. All authors approved the manuscript.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics declarations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe animal study was approved by the Institutional Animal Care and Use Committee of Chonnam National University in Korea (Approval No. CNU IACUC-YB-2022-149).\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eAdditional inquiries can be referred to the corresponding authors; the article contains the original contributions made in the study\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eZeng, A., Li, H., Liu, J. \u0026amp; Wu, M. The Progress of Decellularized Scaffold in Stomatology. \u003cem\u003eTissue Engineering and Regenerative Medicine\u003c/em\u003e\u003cstrong\u003e19,\u003c/strong\u003e 451\u0026ndash;461 (2022).\u003c/li\u003e\n \u003cli\u003ePereira, H. F., Cengiz, I. F., Silva, F. S., Reis, R. L. \u0026amp; Oliveira, J. M. Scaffolds and coatings for bone regeneration. \u003cem\u003eJournal of Materials Science: Materials in Medicine\u003c/em\u003e\u003cstrong\u003e31,\u003c/strong\u003e 27 (2020).\u003c/li\u003e\n \u003cli\u003ePark, J. Y. \u003cem\u003eet al.\u003c/em\u003e 3D printing technology to control BMP-2 and VEGF delivery spatially and temporally to promote large-volume bone regeneration. \u003cem\u003eJournal of Materials Chemistry B\u003c/em\u003e\u003cstrong\u003e3,\u003c/strong\u003e 5415\u0026ndash;5425 (2015).\u003c/li\u003e\n \u003cli\u003eBaldwin, P. \u003cem\u003eet al.\u003c/em\u003e Autograft, Allograft, and Bone Graft Substitutes: Clinical Evidence and Indications for Use in the Setting of Orthopaedic Trauma Surgery. \u003cem\u003eJournal of Orthopaedic Trauma\u003c/em\u003e\u003cstrong\u003e33,\u003c/strong\u003e 203\u0026ndash;213 (2019).\u003c/li\u003e\n \u003cli\u003eZhang, S. \u003cem\u003eet al.\u003c/em\u003e Clinical Reference Strategy for the Selection of Treatment Materials for Maxillofacial Bone Transplantation: A Systematic Review and Network Meta-Analysis. \u003cem\u003eTissue Engineering and Regenerative Medicine\u003c/em\u003e\u003cstrong\u003e19,\u003c/strong\u003e 437\u0026ndash;450 (2022).\u003c/li\u003e\n \u003cli\u003eGenova, T. \u003cem\u003eet al.\u003c/em\u003e Advances on Bone Substitutes through 3D Bioprinting. \u003cem\u003eInternational Journal of Molecular Sciences\u003c/em\u003e\u003cstrong\u003e21,\u003c/strong\u003e 7012 (2020).\u003c/li\u003e\n \u003cli\u003eCancedda, R., Giannoni, P. \u0026amp; Mastrogiacomo, M. A tissue engineering approach to bone repair in large animal models and in clinical practice. \u003cem\u003eBiomaterials\u003c/em\u003e\u003cstrong\u003e28,\u003c/strong\u003e 4240\u0026ndash;4250 (2007).\u003c/li\u003e\n \u003cli\u003eBhattacharyya, A., Khatun, M. R., Narmatha, S., Nagarajan, R. \u0026amp; Noh, I. Modulation of 3D Bioprintability in Polysaccharide Bioink by Bioglass Nanoparticles and Multiple Metal Ions for Tissue Engineering. \u003cem\u003eTissue Engineering and Regenerative Medicine\u003c/em\u003e\u003cstrong\u003e21,\u003c/strong\u003e 261\u0026ndash;275 (2023).\u003c/li\u003e\n \u003cli\u003eFriedenstein, A. J., Chailakhyan, R. K., Latsinik, N. V., Panasyuk, A. F. \u0026amp; Keiliss-Borok, I. V. Stromal cells responsible for transferring the microenvironment of the hemopoietic tissues: cloning in vitro and retransplantation in vivo. \u003cem\u003eTransplantation\u003c/em\u003e\u003cstrong\u003e17,\u003c/strong\u003e 331\u0026ndash;340 (1974).\u003c/li\u003e\n \u003cli\u003eCampagnoli, C. \u003cem\u003eet al.\u003c/em\u003e Identification of mesenchymal stem/progenitor cells in human first-trimester fetal blood, liver, and bone marrow. \u003cem\u003eBlood\u003c/em\u003e\u003cstrong\u003e98,\u003c/strong\u003e 2396\u0026ndash;2402 (2001).\u003c/li\u003e\n \u003cli\u003eErices, A., Conget, P. \u0026amp; Minguell, J. J. Mesenchymal progenitor cells in human umbilical cord blood. \u003cem\u003eBritish Journal of Haematology\u003c/em\u003e\u003cstrong\u003e109,\u003c/strong\u003e 235\u0026ndash;242 (2000).\u003c/li\u003e\n \u003cli\u003eZuk, P. A. \u003cem\u003eet al.\u003c/em\u003e Human Adipose Tissue Is a Source of Multipotent Stem Cells. \u003cem\u003eMolecular Biology of the Cell\u003c/em\u003e\u003cstrong\u003e13,\u003c/strong\u003e 4279\u0026ndash;4295 (2002).\u003c/li\u003e\n \u003cli\u003eYamada, Y. \u003cem\u003eet al.\u003c/em\u003e 3D-cultured small size adipose-derived stem cell spheroids promote bone regeneration in the critical-sized bone defect rat model. \u003cem\u003eBiochemical and Biophysical Research Communications\u003c/em\u003e\u003cstrong\u003e603,\u003c/strong\u003e 57\u0026ndash;62 (2022).\u003c/li\u003e\n \u003cli\u003eReddi, A. H. \u0026amp; Cunningham, N. S. Recent Progress in Bone Induction by Osteogenin and Bone Morphogenetic Proteins: Challenges for Biomechanical and Tissue Engineering. \u003cem\u003eJournal of Biomechanical Engineering\u003c/em\u003e\u003cstrong\u003e113,\u003c/strong\u003e 189\u0026ndash;190 (1991).\u003c/li\u003e\n \u003cli\u003eLee, S., Choi, E., Cha, M.-J. \u0026amp; Hwang, K.-C. Cell Adhesion and Long-Term Survival of Transplanted Mesenchymal Stem Cells: A Prerequisite for Cell Therapy. \u003cem\u003eOxidative Medicine and Cellular Longevity\u003c/em\u003e\u003cstrong\u003e2015,\u003c/strong\u003e 1\u0026ndash;9 (2015).\u003c/li\u003e\n \u003cli\u003eColter, D. C., Class, R., DiGirolamo, C. M. \u0026amp; Prockop, D. J. Rapid expansion of recycling stem cells in cultures of plastic-adherent cells from human bone marrow. \u003cem\u003eProceedings of the National Academy of Sciences\u003c/em\u003e\u003cstrong\u003e97,\u003c/strong\u003e 3213\u0026ndash;3218 (2000).\u003c/li\u003e\n \u003cli\u003eLee, S. I., Ko, Y. \u0026amp; Park, J.-B. Evaluation of the osteogenic differentiation of gingiva-derived stem cells grown on culture plates or in stem cell spheroids: Comparison of two- and three-dimensional cultures. \u003cem\u003eExperimental and Therapeutic Medicine\u003c/em\u003e\u003cstrong\u003e14,\u003c/strong\u003e 2434\u0026ndash;2438 (2017).\u003c/li\u003e\n \u003cli\u003eAchilli, T. M., Meyer, J. \u0026amp; Morgan, J. R. Advances in the formation, use and understanding of multi-cellular spheroids. \u003cem\u003eExpert Opinion on Biological Therapy\u003c/em\u003e\u003cstrong\u003e12,\u003c/strong\u003e 1347\u0026ndash;1360 (2012).\u003c/li\u003e\n \u003cli\u003eLee, D. H. \u0026amp; Bhang, S. H. Development of Hetero-Cell Type Spheroids Via Core\u0026ndash;Shell Strategy for Enhanced Wound Healing Effect of Human Adipose-Derived Stem Cells. \u003cem\u003eTissue Engineering and Regenerative Medicine\u003c/em\u003e\u003cstrong\u003e20,\u003c/strong\u003e 581\u0026ndash;591 (2023).\u003c/li\u003e\n \u003cli\u003eMurphy, K. C., Fang, S. Y. \u0026amp; Leach, J. K. Human mesenchymal stem cell spheroids in fibrin hydrogels exhibit improved cell survival and potential for bone healing. \u003cem\u003eCell and Tissue Research\u003c/em\u003e\u003cstrong\u003e357,\u003c/strong\u003e 91\u0026ndash;99 (2014).\u003c/li\u003e\n \u003cli\u003eBartosh, T. J. \u003cem\u003eet al.\u003c/em\u003e Aggregation of human mesenchymal stromal cells (MSCs) into 3D spheroids enhances their anti-inflammatory properties. \u003cem\u003eProceedings of the National Academy of Sciences\u003c/em\u003e\u003cstrong\u003e107,\u003c/strong\u003e 13724\u0026ndash;13729 (2010).\u003c/li\u003e\n \u003cli\u003eBhang, S. H., Lee, S., Shin, J.-Y., Lee, T.-J. \u0026amp; Kim, B.-S. Transplantation of Cord Blood Mesenchymal Stem Cells as Spheroids Enhances Vascularization. \u003cem\u003eTissue Engineering Part A\u003c/em\u003e\u003cstrong\u003e18,\u003c/strong\u003e 2138\u0026ndash;2147 (2012).\u003c/li\u003e\n \u003cli\u003eRoddy, E., DeBaun, M. R., Daoud-Gray, A., Yang, Y. P. \u0026amp; Gardner, M. J. Treatment of critical-sized bone defects: clinical and tissue engineering perspectives. \u003cem\u003eEuropean Journal of Orthopaedic Surgery \u0026amp;amp, doi:Traumatology\u003c/em\u003e\u003cstrong\u003e28,\u003c/strong\u003e 351\u0026ndash;362 (2017).\u003c/li\u003e\n \u003cli\u003eShen, F. H. \u003cem\u003eet al.\u003c/em\u003e Implications of adipose-derived stromal cells in a 3D culture system for osteogenic differentiation: an in vitro and in vivo investigation. \u003cem\u003eThe Spine Journal\u003c/em\u003e\u003cstrong\u003e13,\u003c/strong\u003e 32\u0026ndash;43 (2013).\u003c/li\u003e\n \u003cli\u003eLee, J.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e Human adipose-derived stem cell spheroids incorporating platelet-derived growth factor (PDGF) and bio-minerals for vascularized bone tissue engineering. \u003cem\u003eBiomaterials\u003c/em\u003e\u003cstrong\u003e255,\u003c/strong\u003e 120192 (2020).\u003c/li\u003e\n \u003cli\u003eLee, J.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e 3D printed micro-chambers carrying stem cell spheroids and pro-proliferative growth factors for bone tissue regeneration. \u003cem\u003eBiofabrication\u003c/em\u003e\u003cstrong\u003e13,\u003c/strong\u003e 015011 (2020).\u003c/li\u003e\n \u003cli\u003eSeol, Y. \u003cem\u003eet al.\u003c/em\u003e A new method of fabricating robust freeform 3D ceramic scaffolds for bone tissue regeneration. \u003cem\u003eBiotechnology and Bioengineering\u003c/em\u003e\u003cstrong\u003e110,\u003c/strong\u003e 1444\u0026ndash;1455 (2013).\u003c/li\u003e\n \u003cli\u003eYeo, A., Sju, E., Rai, B. \u0026amp; Teoh, S. H. Customizing the degradation and load‐bearing profile of 3D polycaprolactone‐tricalcium phosphate scaffolds under enzymatic and hydrolytic conditions. \u003cem\u003eJournal of Biomedical Materials Research Part B: Applied Biomaterials\u003c/em\u003e\u003cstrong\u003e87B,\u003c/strong\u003e 562\u0026ndash;569 (2008).\u003c/li\u003e\n \u003cli\u003eLiu, N., Zhang, X., Guo, Q., Wu, T. \u0026amp; Wang, Y. 3D Bioprinted Scaffolds for Tissue Repair and Regeneration. \u003cem\u003eFront. Mater.\u003c/em\u003e\u003cstrong\u003e9,\u0026nbsp;\u003c/strong\u003e925321 (2022).\u003c/li\u003e\n \u003cli\u003eZhang, W.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e Fabrication and characterization of porous polycaprolactone scaffold via extrusion-based cryogenic 3D printing for tissue engineering. \u003cem\u003eMaterials \u0026amp;amp; Design\u003c/em\u003e\u003cstrong\u003e180,\u003c/strong\u003e 107946 (2019).\u003c/li\u003e\n \u003cli\u003eSiddiqui, N., Asawa, S., Birru, B., Baadhe, R. \u0026amp; Rao, S. PCL-Based Composite Scaffold Matrices for Tissue Engineering Applications. \u003cem\u003eMol Biotechnol\u003c/em\u003e\u003cstrong\u003e60,\u003c/strong\u003e 506\u0026ndash;532 (2018).\u003c/li\u003e\n \u003cli\u003eRosales-Ib\u0026aacute;\u0026ntilde;ez, R.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e Assessment of a PCL-3D Printing-Dental Pulp Stem Cells Triplet for Bone Engineering: An In Vitro Study. \u003cem\u003ePolymers\u003c/em\u003e\u003cstrong\u003e13,\u003c/strong\u003e 1154 (2021).\u003c/li\u003e\n \u003cli\u003eJiao, Z.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e 3D printing of HA / PCL composite tissue engineering scaffolds. \u003cem\u003eAdvanced Industrial and Engineering Polymer Research\u003c/em\u003e\u003cstrong\u003e2,\u003c/strong\u003e 196\u0026ndash;202 (2019).\u003c/li\u003e\n \u003cli\u003eKim, C. G. \u003cem\u003eet al.\u003c/em\u003e Fabrication of Biocompatible Polycaprolactone\u0026ndash;Hydroxyapatite Composite Filaments for the FDM 3D Printing of Bone Scaffolds. \u003cem\u003eApplied Sciences\u003c/em\u003e\u003cstrong\u003e11,\u003c/strong\u003e 6351 (2021).\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eJEONG, K. J. \u003cem\u003eet al.\u003c/em\u003e Successful Clinical Application of Cancellous Allografts With Structural Support for Failed Bone Fracture Healing in Dogs. \u003cem\u003eIn Vivo\u003c/em\u003e\u003cstrong\u003e33,\u003c/strong\u003e 1813\u0026ndash;1818 (2019).\u003c/li\u003e\n \u003cli\u003eShanbhag, S.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e Bone regeneration in rat calvarial defects using dissociated or spheroid mesenchymal stromal cells in scaffold-hydrogel constructs. \u003cem\u003eStem Cell Res Ther\u003c/em\u003e\u003cstrong\u003e12,\u0026nbsp;\u003c/strong\u003e575 (2021).\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eKronemberger, G. S.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e A Synergic Strategy: Adipose-Derived Stem Cell Spheroids Seeded on 3D-Printed PLA/CHA Scaffolds Implanted in a Bone Critical-Size Defect Model. \u003cem\u003eJFB\u003c/em\u003e\u003cstrong\u003e14,\u003c/strong\u003e 555 (2023).\u003c/li\u003e\n \u003cli\u003eKusuma, G. D., Carthew, J., Lim, R. \u0026amp; Frith, J. E. Effect of the microenvironment on mesenchymal stem cell paracrine signaling: opportunities to engineer the therapeutic effect. \u003cem\u003eStem Cells Dev\u003c/em\u003e\u003cstrong\u003e26,\u0026nbsp;\u003c/strong\u003e617-631 (2017)\u0026nbsp;\u003c/li\u003e\n \u003cli\u003ePotapova, I. A. \u003cem\u003eet al\u003c/em\u003e. Mesenchymal stem cells support migration, extracellular matrix invasion, proliferation, and survival of endothelial cells in vitro. \u003cem\u003eStem Cells\u003c/em\u003e\u003cstrong\u003e25,\u003c/strong\u003e 1761-1768 (2007)\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eZhao, N. \u003cem\u003eet al.\u003c/em\u003e Exogenous signaling\u0026nbsp;molecules released from aptamer-functionalized hydrogels promote the survival of mesenchymal stem cell spheroids. \u003cem\u003eACS Appl. Mater. Interfaces\u003c/em\u003e\u003cstrong\u003e12,\u003c/strong\u003e 24599-24610 (2020)\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eJafary, F., Hanachi, P. \u0026amp; Gorjipour, K. Osteoblast Differentiation on Collagen Scaffold with Immobilized Alkaline Phosphatase. \u003cem\u003eInt J Organ Transplant Med\u0026nbsp;\u003c/em\u003e\u003cstrong\u003e8,\u0026nbsp;\u003c/strong\u003e195-202 (2017).\u003c/li\u003e\n \u003cli\u003eHashemibeni, B.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e Comparison of Phenotypic Characterization between Differentiated Osteoblasts from Stem Cells and Calvaria Osteoblasts In vitro. \u003cem\u003eInt J Prev Med\u0026nbsp;\u003c/em\u003e\u003cstrong\u003e4,\u0026nbsp;\u003c/strong\u003e180-186 (2013).\u003c/li\u003e\n \u003cli\u003eGregory, C. A., Grady Gunn, W., Peister, A. \u0026amp; Prockop, D. J. An Alizarin red-based assay of mineralization by adherent cells in culture: comparison with cetylpyridinium chloride extraction. \u003cem\u003eAnalytical Biochemistry\u003c/em\u003e\u003cstrong\u003e329,\u003c/strong\u003e 77\u0026ndash;84 (2004).\u003c/li\u003e\n \u003cli\u003eKim, Y. B. \u0026amp; Kim, G. H. PCL/Alginate Composite Scaffolds for Hard Tissue Engineering: Fabrication, Characterization, and Cellular Activities. \u003cem\u003eACS Comb. Sci.\u003c/em\u003e\u003cstrong\u003e17,\u003c/strong\u003e 87\u0026ndash;99 (2015).\u003c/li\u003e\n \u003cli\u003eLangenbach, F. \u003cem\u003eet al\u003c/em\u003e. Scaffold-free microtissues: differences from monolayer cultures and their potential in bone tissue engineering. \u003cem\u003eClin Oral Investig\u0026nbsp;\u003c/em\u003e\u003cstrong\u003e17,\u003c/strong\u003e 9-17 (2013).\u003c/li\u003e\n \u003cli\u003eLi, N.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e Spontaneous spheroids from alveolar bone-derived mesenchymal stromal cells maintain pluripotency of stem cells by regulating hypoxia-inducible factors. \u003cem\u003eBiol Res\u003c/em\u003e\u003cstrong\u003e56,\u0026nbsp;\u003c/strong\u003e17 (2023).\u003c/li\u003e\n \u003cli\u003eGurumurthy, B., Bierdeman, P. C. \u0026amp; Janorkar, A. V. Spheroid model for functional osteogenic evaluation of human adipose derived stem cells. \u003cem\u003eJ Biomedical Materials Res\u003c/em\u003e\u003cstrong\u003e105,\u003c/strong\u003e 1230\u0026ndash;1236 (2017).\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eVillalona, G. A.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e Cell-Seeding Techniques in Vascular Tissue Engineering. \u003cem\u003eTissue Engineering Part B: Reviews\u003c/em\u003e\u003cstrong\u003e16,\u003c/strong\u003e 341\u0026ndash;350 (2010).\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eHo, S. S., Hung, B. P., Heyrani, N., Lee, M. A. \u0026amp; Leach, J. K. Hypoxic Preconditioning of Mesenchymal Stem Cells with Subsequent Spheroid Formation Accelerates Repair of Segmental Bone Defects. \u003cem\u003eStem Cells\u003c/em\u003e\u003cstrong\u003e36,\u003c/strong\u003e 1393\u0026ndash;1403 (2018).\u003c/li\u003e\n \u003cli\u003eHo, S. S., Keown, A. T., Addison, B. \u0026amp; Leach, J. K. Cell Migration and Bone Formation from Mesenchymal Stem Cell Spheroids in Alginate Hydrogels Are Regulated by Adhesive Ligand Density. \u003cem\u003eBiomacromolecules\u003c/em\u003e\u003cstrong\u003e18,\u003c/strong\u003e 4331\u0026ndash;4340 (2017).\u003c/li\u003e\n \u003cli\u003eHo, S. S., Murphy, K. C., Binder, B. Y. K., Vissers, C. B. \u0026amp; Leach, J. K. Increased Survival and Function of Mesenchymal Stem Cell Spheroids Entrapped in Instructive Alginate Hydrogels. \u003cem\u003eStem Cells Translational Medicine\u003c/em\u003e\u003cstrong\u003e5,\u003c/strong\u003e 773\u0026ndash;781 (2016).\u003c/li\u003e\n \u003cli\u003eLee, C. F. \u003cem\u003eet al.\u003c/em\u003e 3D Printing of Collagen/Oligomeric Proanthocyanidin/Oxidized Hyaluronic Acid Composite Scaffolds for Articular Cartilage Repair. \u003cem\u003ePolymers\u003c/em\u003e\u003cstrong\u003e13,\u003c/strong\u003e 3123 (2021).\u003c/li\u003e\n \u003cli\u003eJayatissa, N. U. \u0026amp; Bhaduri, S. 3D Printed Polymer Scaffolds for Bone Tissue Regeneration. \u003cem\u003eJ Emerg Invest\u0026nbsp;\u003c/em\u003e\u003cstrong\u003e26,\u0026nbsp;\u003c/strong\u003e2\u0026nbsp;(2019).\u003c/li\u003e\n \u003cli\u003eMarouf, N., Nojehdehian, H. \u0026amp; Ghorbani, F. Physicochemical properties of chitosan\u0026ndash;hydroxyapatite matrix incorporated with \u003cem\u003eGinkgo biloba\u003c/em\u003e-loaded PLGA microspheres for tissue engineering applications. \u003cem\u003ePolymers and Polymer Composites\u003c/em\u003e\u003cstrong\u003e28,\u003c/strong\u003e 320\u0026ndash;330 (2019).\u003c/li\u003e\n \u003cli\u003eLi, H., Tan, C. \u0026amp; Li, L. Review of 3D printable hydrogels and constructs. \u003cem\u003eMaterials \u0026amp;amp; Design\u003c/em\u003e\u003cstrong\u003e159,\u003c/strong\u003e 20\u0026ndash;38 (2018).\u003c/li\u003e\n \u003cli\u003eWang, X. \u003cem\u003eet al.\u003c/em\u003e Three-Dimensional, MultiScale, and Interconnected Trabecular Bone Mimic Porous Tantalum Scaffold for Bone Tissue Engineering. \u003cem\u003eACS Omega\u003c/em\u003e\u003cstrong\u003e5,\u003c/strong\u003e 22520\u0026ndash;22528 (2020).\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eMa, P. X. Biomimetic materials for tissue engineering. \u003cem\u003eAdvanced Drug Delivery Reviews\u003c/em\u003e\u003cstrong\u003e60,\u003c/strong\u003e 184\u0026ndash;198, (2008).\u003c/li\u003e\n \u003cli\u003eDeshpande, M. V., Girase, A. \u0026amp; King, M. W. Degradation of Poly(\u0026epsilon;-caprolactone) Resorbable Multifilament Yarn under Physiological Conditions. \u003cem\u003ePolymers\u003c/em\u003e\u003cstrong\u003e15,\u003c/strong\u003e 3819 (2023).\u003c/li\u003e\n \u003cli\u003eJo, H. M.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e Application of modified porcine xenograft by collagen coating in the veterinary field: pre-clinical and clinical evaluations. \u003cem\u003eFront. Vet. Sci.\u003c/em\u003e\u003cstrong\u003e11,\u0026nbsp;\u003c/strong\u003e1373099 (2024).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"critical-size defect, bone tissue engineering, spheroids, synergic strategy, 3D-printed scaffold, bone regeneration","lastPublishedDoi":"10.21203/rs.3.rs-6291864/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6291864/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eBone tissue is generally resilient and can self-heal, but critical-size defects (CSDs) with complex geometries cannot be repaired without clinical intervention. Customized scaffolds developed using three-dimensional (3D) printing techniques can effectively repair complex-shaped CSDs. Adipose-derived stem cells (ADSCs), a type of mesenchymal stem cell (MSC), can differentiate into osteoblasts and exhibit osteoinductive properties. However, ADSC-single cells fabricated via two-dimensional (2D) monolayer cultures have limitations in maintaining cell survival and function over time. Unlike 2D monolayer cultures, ADSC-spheroids fabricated via 3D spheroid cultures can overcome this limitation by increasing the survival of ADSCs and enhancing their \u003cem\u003ein vivo\u003c/em\u003e osteogenic capacity. This study aimed to evaluate the potential of a synergistic strategy of ADSC-spheroids within a 3D-printed scaffold made of polycaprolactone/hydroxyapatite (PCL/HA) in bone regeneration. \u003cem\u003eIn vitro\u003c/em\u003e experiments demonstrated that ADSC-spheroids promoted mineralization in 3D-printed scaffolds. Radiographs and histological analysis performed at eight weeks post-implantation in \u003cem\u003ein vivo\u003c/em\u003e experiments using a rabbit radial defect model showed successful bone regeneration in the group containing ADSC-spheroids within the PCL/HA scaffold. These results suggest that the synergistic strategy of incorporating ADSC-spheroids into 3D-printed PCL/HA scaffolds shows promise for clinical applications in treating complex CSDs.\u003c/p\u003e","manuscriptTitle":"Bone regeneration using stem cell spheroids within 3D-printed scaffolds in a rabbit radial defect model","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-25 15:30:30","doi":"10.21203/rs.3.rs-6291864/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-05-12T16:17:15+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-05-09T15:34:56+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"35720016038870056888805868536497764730","date":"2025-04-30T15:03:09+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-04-18T02:20:10+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"262162854560726542727001856754398341077","date":"2025-04-09T02:25:47+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"176254211420029278982373914462978296224","date":"2025-04-04T15:16:16+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-04-04T12:39:53+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-04-04T12:31:56+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-04-03T14:49:56+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-04-02T10:39:35+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-03-24T05:26:00+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"498b2054-6950-4277-b06c-0332a7c1c4ab","owner":[],"postedDate":"April 25th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":47340144,"name":"Biological sciences/Biotechnology/Biomaterials/Biomedical materials"},{"id":47340145,"name":"Biological sciences/Biotechnology/Biomaterials/Tissues"}],"tags":[],"updatedAt":"2025-12-22T16:11:40+00:00","versionOfRecord":{"articleIdentity":"rs-6291864","link":"https://doi.org/10.1038/s41598-025-25581-5","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2025-12-16 15:57:22","publishedOnDateReadable":"December 16th, 2025"},"versionCreatedAt":"2025-04-25 15:30:30","video":"","vorDoi":"10.1038/s41598-025-25581-5","vorDoiUrl":"https://doi.org/10.1038/s41598-025-25581-5","workflowStages":[]},"version":"v1","identity":"rs-6291864","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6291864","identity":"rs-6291864","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}