Bio3D graft engineered from human mesenchymal stromal cells/extracellular complexes facilitate periodontal tissue regeneration

Bio3D graft engineered from human mesenchymal stromal cells/extracellular complexes facilitate periodontal tissue regeneration | 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 Bio3D graft engineered from human mesenchymal stromal cells/extracellular complexes facilitate periodontal tissue regeneration Hiroki Yoshii, Mai Yoshino, Masayuki Suzuki, Hisakatsu Sone, Tetsuya Yoshimoto, and 9 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7953573/v1 This work is licensed under a CC BY 4.0 License Status: Under Revision Version 1 posted 9 You are reading this latest preprint version Abstract Periodontitis, a chronic inflammatory disease leading to irreversible loss of tooth-supporting tissues, remains a major unmet clinical challenge despite advances in biomaterial-based cell therapies. Scaffold-free regenerative strategies that fully rely on cellular and extracellular matrix (ECM) components may overcome the limitations of artificial scaffolds. Here, we developed a human Bio3D graft fabricated from clumps of mesenchymal stromal cells and their self-produced ECM (C-MSCs) using a Bio3D printer. When transplanted into a critical-size periodontal defect in immunodeficient rats, the Bio3D graft achieved safe and reproducible regeneration of cementum, periodontal ligament, and alveolar bone without ectopic or excessive bone formation. Transcriptomic and histological analyses revealed enhanced YAP/TAZ signaling in the Bio3D graft compared with C-MSCs, and functional inhibition of YAP/TAZ abolished periodontal reconstruction, demonstrating its essential role. These findings establish a foundation for next-generation, scaffold-free cell therapies capable of reconstructing complex periodontal structures. Biological sciences/Biotechnology Biological sciences/Cell biology Physical sciences/Materials science Health sciences/Medical research Biological sciences/Stem cells Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Periodontitis is a chronic inflammatory disease caused by the host immune response to bacterial infection, leading to the progressive destruction of periodontal tissues, including cementum, periodontal ligament, and alveolar bone 1 , 2 . Without appropriate treatment, this breakdown ultimately results in tooth loss. Moreover, an increasing body of evidence suggests that periodontitis and tooth loss contribute to the exacerbation of various systemic diseases, such as dementia, pneumonia, diabetes, cardiovascular disease, and hepatitis 3 – 5 . Therefore, establishing a curative therapy that enables true periodontal tissue regeneration is crucial for extending healthy life expectancy in aging societies. Mesenchymal stromal/stem cells (MSCs), originally identified in the bone marrow, contribute to tissue homeostasis through their multipotent differentiation and self-renewal capabilities 6 , 7 . Consequently, regenerative therapies using MSCs have attracted considerable medical and scientific attention over the past two decades 8 . In the context of periodontal regeneration, numerous preclinical and clinical studies have explored autologous MSC transplantation combined with biomaterial scaffolds 9 , 10 . While these strategies have demonstrated promising outcomes, their clinical efficacy in humans remains variable and often suboptimal 11 – 13 . One critical limitation lies in the use of artificial scaffolds, which, as foreign materials, are not inherently compatible with the regeneration of authentic periodontal tissue. Successful regeneration requires that such scaffolds be gradually degraded and replaced by host-derived periodontal components—a process that can delay healing and introduce risks of bacterial infection and adverse inflammatory responses. To overcome these challenges, we previously developed a scaffold-free transplantation method using clumps of MSCs and their self-produced extracellular matrix (C-MSCs). Unlike small MSC spheroids (typically < 500 µm) that are generated predominantly through cell-cell contacts, C-MSCs are approximately 1 mm in diameter and are composed of self-produced type I collagen 14 —the principal extracellular matrix component of periodontal and bone tissues—and can be transplanted directly into defects without the need for artificial materials 15 , 16 . C-MSCs not only eliminate the necessity for scaffold degradation but also provide an intrinsic biological microenvironment that supports cell viability, site-specific differentiation, and periodontal tissue and calvaria bone reconstruction 17 , 18 . In addition, the functional properties of C-MSCs, including ECM composition, osteogenic potential, and immunomodulatory capacity, can be modulated in vitro by altering culture conditions prior to transplantation 19 – 21 . To further advance this approach toward clinical application, improvements in graft size, handling properties, and functional performance were required. In this study, we addressed these needs by employing Bio3D printing technology 22 to fabricate a novel, large-sized, scaffold-free MSC construct, which we termed the Bio3D graft. This construct was generated by skewering individual C-MSCs onto a fine needle-array using a Bio3D printer, allowing the clumps to fuse into a cohesive three-dimensional tissue-like structure. This needle-array-based assembly technique enables precise control of graft shape and size while maintaining a fully scaffold-free composition composed entirely of viable MSCs and their endogenous extracellular matrix. To establish non-clinical proof of concept (PoC) for periodontal tissue regenerative therapy using the Bio3D graft, we fabricated human Bio3D grafts by combining human bone marrow-derived MSCs with the clump culture method and Bio3D printing technology. The generated grafts were transplanted into a nude rat periodontal dehiscence defect model, which represents one of the largest standardized-size critical defects in immunodeficient animals. We further analyzed the unique biological characteristics of the Bio3D grafts and investigated the underlying mechanisms of tissue regeneration. Compared to conventional C-MSCs, which are limited to small spherical clumps (~ 1 mm), the Bio3D graft exhibited a structurally robust, large-scale architecture that allowed easy transplantation into large defects without the need for artificial scaffolds. The graft consistently induced reliable periodontal tissue regeneration. Notably, the Bio3D grafts demonstrated enhanced YAP/TAZ signaling activity, which facilitated appropriate differentiation of the transplanted human cells and led to successful reconstruction of periodontal tissues. Taken together, the present study highlights the potential of the Bio3D graft as an innovative, scaffold-free regenerative approach in which MSCs self-organize into large, clinically applicable implants capable of reconstructing complex periodontal tissues. Results Generation of human Bio3D grafts tailored to the nude rat periodontal dehiscence defect model We first generated clumps of mesenchymal stromal cells and extracellular matrix (C-MSCs) approximately 1.2 mm in diameter from human bone marrow-derived MSCs, as described in the Materials and Methods (Fig. 1 A (a,b)). Histological and immunofluorescence analyses revealed that C-MSCs consisted of viable cells embedded within a self-produced type I collagen (COL1) matrix (Fig. 1 B, 1 C, upper panels). Previously, it was demonstrated that transplantation of multiple C-MSCs without artificial scaffolds into a 2 mm × 3 mm × 1 mm rat periodontal fenestration defect 23 —an accessible defect model in which the cervical area of alveolar bone is preserved—successfully induced periodontal tissue regeneration 18 . To determine whether scaffold-free C-MSCs transplantation could also be effective in a more challenging setting, we transplanted twelve human C-MSCs into a 4 mm × 3 mm × 1 mm nude rat periodontal dehiscence defect model developed as a modification of the fenestration defect model, in which the cervical bone is completely removed, creating a standardized-size critical defect (Fig. 1 D). This dehiscence model mimics severe periodontitis with direct communication between the defect and the oral cavity. However, C-MSCs transplantation failed to induce effective periodontal tissue regeneration compared with the no-graft control (Supplementary Fig. 1), possibly due to insufficient engraftment of transplanted C-MSCs in the absence of cervical bone support and the inability of small clumps to exert adequate regenerative function within such a large defect. We therefore sought to fabricate a Bio3D graft precisely matching the geometry of the nude rat dehiscence defect, enabling stable engraftment and more effectively exerting cellular functions. To this end, C-MSCs were assembled using a Bio3D printer by stacking them on a fine needle-array in a 4 × 4 arrangement, allowing them to fuse into a single construct (Fig. 1 A (c)). After eight days of culture, this process yielded a structurally stable approximately 3–4 mm square tissue construct, hereafter referred to as the Bio3D graft (Fig. 1 A (d)). Histological examination confirmed that, similar to the original C-MSCs, the Bio3D graft was composed of viable cells embedded in a COL1 matrix. As anticipated, the fabricated Bio3D graft could be easily placed directly into the 4 mm × 3 mm × 1 mm dehiscence defect without the need for artificial materials, achieving full coverage of the defect site (Fig. 1 E). Human Bio3D graft induces successful periodontal tissue regeneration in a nude rat dehiscence defect model Since the human Bio3D graft could be successfully placed into the standardized-size nude rat periodontal dehiscence defect (Fig. 1 ), we next monitored its effect on alveolar bone regeneration using micro-computed tomography (micro-CT). In the no-graft group, minimal bone formation was observed up to 4 weeks post-surgery, and even at 8 weeks the defect remained largely unhealed, with persistent root exposure (Fig. 2 A). In contrast, in the Bio3D graft group, early bone formation was evident early as 2 weeks post-transplantation. By 4 weeks, regenerated bone fully covered the previously exposed root surfaces, and by 8 weeks the defect was completely filled with mature bone (Fig. 2 A, 2 C left graph). Notably, sagittal and horizontal micro-CT images at 8 weeks revealed a radiolucent space between the root surface and the newly formed bone (red arrows in Fig. 2 A), suggesting preservation of a functional periodontal ligament space without ankylosis (Fig. 2 A, lower right panels). To determine whether the Bio3D graft could induce appropriate regeneration of all periodontal components—cementum, periodontal ligament, and alveolar bone—we performed histological analyses using HE staining. Consistent with the CT findings, the no-graft group at 8 weeks exhibited limited periodontal tissue, with the defect site largely occupied by granulation tissue-like soft connective tissue (Fig. 2 B). In contrast, by 4 weeks post-transplantation, the Bio3D graft group demonstrated periodontal tissue regeneration covering the root surface. At 8 weeks, high-magnification views clearly showed a periodontal ligament structure connecting the newly formed alveolar bone and newly deposited cementum along the root surface, particularly in the coronal region near the top of the defect. For quantitative assessment of functional periodontal regeneration, we measured the length of newly formed cementum. The Bio3D graft group showed a significant increase in cementum formation compared with the no-graft group (Fig. 2 C right graph). Collectively, these results indicate that the human Bio3D graft effectively promotes regeneration of functional periodontal tissues in a critical-size nude rat dehiscence defect model. Transplanted human donor cells contribute to periodontal tissue regeneration induced by the Bio3D graft. To investigate the role of transplanted human cells in Bio3D graft–induced periodontal regeneration, we performed immunofluorescence staining with an anti-human vimentin antibody to distinguish between donor human cells and host rat cells. At 2 weeks post-surgery, abundant human vimentin–positive cells descended from the Bio3D graft were detected within the defect area, including in regions corresponding to newly forming alveolar bone and the periodontal ligament space at the bottom of the defect (Fig. 3 A (a,b)). Notably, human cells were also observed in regenerated periodontal tissues—comprising cementum, ligament space, and alveolar bone—even at later time points of 4 and 8 weeks post-transplantation (Fig. 3 A (c-f)). At 8 weeks, human cells persisted not only within the regenerated periodontal ligament space but also along the periosteal surface of the matured alveolar bone. To assess whether the donor human cells within regenerated periodontal tissue had undergone appropriate lineage differentiation, we conducted immunostaining for periostin (POSTN), a marker of periodontal ligament fibroblasts 24 . Multiplex staining demonstrated that many human vimentin–positive cells located in the regenerated ligament space co-expressed periostin (Fig. 3 B). In agreement with the known expression of periostin by periosteal cells 24 , human cells at the periosteal surface of the regenerated alveolar bone also co-expressed periostin, appearing as yellow signals in merged images (Fig. 3 B). We next quantified the number of donor-derived human cells in the regenerated tissues over time. The number of human cells decreased progressively from 2 to 8 weeks post-transplantation (Fig. 3 C). By 8 weeks, within the regenerated ligament space, the number of human cells and host rat cells were comparable; however, in the regenerated alveolar bone, host cells were significantly more abundant than human cells (Fig. 3 D). These observations suggest that Bio3D graft–derived human MSCs initially differentiate into periodontal component cells and contribute directly to tissue reconstruction, and that over time, these regenerated tissues may undergo normal physiological turnover, gradually becoming populated predominantly by host-derived cells while maintaining a functional and stable periodontal architecture. Fabrication of Bio3D grafts using Bio3D printing enhances YAP/TAZ activity For clinical translation of Bio3D grafts, it is essential to understand their cellular characteristics precisely. We therefore performed bulk RNA sequencing to compare the transcriptomic profiles of the Bio3D grafts with those of the original C-MSCs used as the starting material. Differential expression analysis identified 1,245 genes that were upregulated more than 1.5-fold in the Bio3D grafts relative to C-MSCs, whereas 614 genes were downregulated (Fig. 4 A). Gene Ontology enrichment analysis of the upregulated genes revealed significant enrichment for terms related to collagen-containing extracellular matrix and actin filament bundle (Fig. 4 B). To predict transcriptional factors potentially responsible for these changes, we queried the ChEA3 database. Notably, among the top four ranked transcriptional regulators for each term, five genes were known targets or interacting partners of the transcriptional co-activators YAP/TAZ (highlighted in red in Fig. 4 C) 25 – 29 . It is well accepted that YAP/TAZ are key mechanotransducers in MSCs to control cell fate 30 . More specifically, increased YAP/TAZ activity promotes MSCs differentiation toward osteogenic and periodontal tissue–forming lineages 31 , 32 . We therefore examined the expression levels of YAP/TAZ themselves, their downstream target genes, and genes associated with osteogenesis and periodontal tissue formation. As anticipated, these genes were markedly upregulated in Bio3D grafts compared with C-MSCs (Fig. 4 D). The mechanotransducer YAP/TAZ shuttle between the cytoplasm and nucleus, with nuclear localization indicative of active signaling 33 . Thus, to validate the RNA-seq findings at the protein level, we performed immunofluorescence staining for YAP/TAZ. In C-MSCs, YAP/TAZ predominantly localized in the cytoplasm, consistent with low activity. By contrast, a larger proportion of cells in the Bio3D grafts exhibited nuclear localization of YAP/TAZ, indicating enhanced pathway activation (Fig. 4 E,F). Besides, the levels of CTGF production, a well-established downstream target of YAP/TAZ signaling, was significantly increased in Bio3D grafts compared to C-MSCs (Fig. 4 G). Collectively, these results demonstrate that Bio3D grafts, fabricated from C-MSCs by Bio3D printing system, not only increase construct size but also enhance YAP/TAZ activity, a mechanotransduction pathway known to facilitate osteogenic and periodontal tissue–related differentiation. YAP/TAZ activity induced by the Bio3D printing process is required for Bio3D graft–mediated periodontal tissue regeneration Finally, to examine whether the YAP/TAZ activity induced by the Bio3D printing process contributes to the periodontal regenerative capacity of Bio3D grafts, we conducted an inhibition experiment. Briefly, C-MSCs were assembled on the needle array of the Bio3D printer to fabricate Bio3D grafts in the presence or absence of YAP/TAZ inhibitor II (Fig. 5 A). Treatment with the inhibitor did not cause any observable difference in graft size (Fig. 5 B) or degradation of extracellular matrix proteins (Supplementary Fig. 2A); however, it clearly suppressed YAP/TAZ activity, as evidenced by cytoplasmic translocation and degradation of YAP/TAZ (Supplementary Fig. 2B) and reduction in CTGF (Supplementary Fig. 2C). These results confirmed that Bio3D grafts generated with YAP/TAZ-specific inhibitor exhibited diminished YAP/TAZ activity. The inhibitor-treated Bio3D grafts were then transplanted into a nude rat periodontal dehiscence defect model. Notably, micro-CT analysis at 4 weeks post-transplantation demonstrated that defects receiving inhibitor-treated grafts showed no appreciable alveolar bone regeneration (Fig. 5 C, D). In agreement with this finding, histological analysis revealed that inhibitor-treated grafts failed to reconstruct the periodontium—comprising cementum, periodontal ligament, and alveolar bone—and instead formed only fibrous connective tissue (Fig. 5 E, F). To assess the persistence of donor human cells after transplantation, immunofluorescence staining for human vimentin was performed. Importantly, the number of vimentin-positive human cells engrafted within the fibrous tissue of defects receiving inhibitor-treated grafts was comparable to that in defects receiving untreated grafts (Fig. 5 G, H). Collectively, these findings indicate that although human cells from inhibitor-treated Bio3D grafts can engraft within the defect, suppression of YAP/TAZ activity may prevent appropriate lineage differentiation and subsequent reconstruction of periodontal tissues. Thus, YAP/TAZ activity constitutes an intrinsic cellular property of Bio3D grafts that is essential for their periodontal regenerative function. Discussion We previously developed a scaffold-free cell transplantation strategy using clumps of MSCs and their extracellular matrix proteins (C-MSCs), approximately 1 mm in diameter, which enabled bone and periodontal tissue regeneration without artificial materials 15 , 16 , 18 . In the present study, we advanced this approach by employing the Bio3D printer Regenova® to assemble C-MSCs on a needle array and fabricate larger human Bio3D grafts with enhanced regenerative capacity. Importantly, the Bio3D grafts were not only increased in size but also exhibited elevated YAP/TAZ activity, which proved to be functionally required for their superior periodontal regenerative effects. Thus, fabrication of Bio3D grafts by Bio3D printing improves not only the handling and structural adaptability of the grafts to fit complex periodontal defects, but also enhances their intrinsic cellular properties. Collectively, these findings highlight human Bio3D grafts as an innovative scaffold-free cell therapy that combines operability with improved functionality for periodontal tissue regeneration. Previous studies have suggested that paracrine effects, rather than direct differentiation, are primarily responsible for periodontal tissue regeneration induced by grafted MSCs 34 – 36 . In contrast, our findings indicate that, following Bio3D graft transplantation, donor human cells themselves participated in periodontal tissue reconstruction (Fig. 3 ). Specifically, cementum, periodontal ligament, and alveolar bone were regenerated through appropriate lineage differentiation of the transplanted human MSCs. This direct contribution may be explained by the unique scaffold-free architecture of the Bio3D graft. Because the graft is composed of MSCs embedded within their self-produced ECM proteins, particularly COL1 (Fig. 1 ), it could provide a biologically native microenvironment at the defect site without introducing artificial scaffolds that could act as foreign bodies. Compared with conventional artificial scaffold-based MSC transplantation, this environment likely facilitated the intrinsic multipotency of MSCs, thereby promoting site-specific differentiation and tissue reconstruction by the transplanted cells themselves. In particular, as COL1 is the predominant ECM protein in bone and periodontal ligament 37 , 38 , Bio3D grafts enriched in COL1 may provide a favorable cellular microenvironment for grafted MSCs to differentiate into cementoblasts, periodontal ligament fibroblasts, and osteoblasts. Although further investigation is needed to identify the specific environmental cues that direct MSCs toward these periodontal lineages, our findings highlight that Bio3D graft–mediated regeneration involves not only paracrine signaling but also a substantive contribution from the intrinsic multipotency of MSCs. This perspective revisits a classical concept of MSC-based regenerative therapy—tissue reconstruction through differentiation of transplanted cells—which has often been overshadowed by the paracrine paradigm in recent years 39 , 40 . In this study, the number of transplanted human MSCs within the defect gradually decreased over time, and importantly, no evidence of bone overgrowth, ankylosis, or ectopic tissue formation was observed at 8 weeks after surgery. These findings may support the safety of periodontal regenerative therapy using human Bio3D grafts and represent a favorable outcome from a translational perspective. At the same time, it is noteworthy that at 8 weeks post-transplantation, numerous donor-derived human cells expressing POSTN persisted within the newly formed periodontal ligament space and along the periosteal surface of alveolar bone. POSTN is highly enriched in the periodontal ligament and is indispensable for maintaining the periodontal homeostasis 41 , 42 . In addition, POSTN-positive stromal stem/progenitor cells in the periosteum contribute to bone homeostasis and regeneration 43 , 44 . Thus, although the majority of transplanted cells likely disappeared after participating in tissue reconstruction through normal turnover, those that reached the stem cell niches of the periodontal ligament and periosteum may have persisted and contributed to long-term maintenance of tissue integrity. Taken together, these findings suggest that Bio3D graft transplantation can achieve safe and reliable functional regeneration of periodontal tissues without undesirable outcomes such as ectopic tissue formation or tumorigenesis, while supporting long-term maintenance of tissue homeostasis. Human Bio3D grafts fabricated from C-MSCs using the Bio3D printer Regenova® exhibited markedly elevated YAP/TAZ activity (Fig. 4 ). In MSCs, increased YAP/TAZ signaling is well known to promote lineage commitment toward osteogenic 45 and periodontal tissue–forming cells 46 . Thus, the reconstruction of cementum, periodontal ligament, and alveolar bone by transplanted human MSCs observed in this study is most likely attributable to enhanced YAP/TAZ activity. This interpretation is strongly supported by the finding that Bio3D grafts generated in the presence of a YAP/TAZ inhibitor retained engrafted human cells within the defect but failed to induce periodontal tissue reconstruction (Fig. 5 ), indicating that suppression of YAP/TAZ activity impaired appropriate differentiation. The precise molecular mechanisms responsible for the elevated YAP/TAZ activity in Bio3D grafts were not clarified in this study. However, YAP/TAZ are established mechanotransducers that convert substrate stiffness and physical forces into biochemical signals 47 . In particular, abundant ECM deposition, increased matrix rigidity, or mechanical loading are well known to activate YAP/TAZ and direct MSCs toward osteogenic differentiation 48 . It is therefore plausible that the Bio3D fabrication process—including the stacking of C-MSCs on the needle array, the sensing of needle stiffness and the presence of abundant COL1 matrix—collectively provided mechanical cues that enhanced YAP/TAZ activity. Because YAP/TAZ are transcriptional co-activators that shuttle between the cytoplasm and nucleus, their activity is often assessed indirectly by measuring the expression of downstream target genes. Among these, connective tissue growth factor (CTGF/CCN2) is a particularly reliable surrogate marker, as its expression closely reflects YAP/TAZ activity 20 , 27 , 30 . Consistent with this, our study demonstrated that CTGF levels in the culture supernatant provided a convenient readout of YAP/TAZ activity in Bio3D grafts. Importantly, from a translational perspective, CTGF production not only reflects YAP/TAZ signaling but also correlates with the regenerative potency of Bio3D grafts, highlighting its potential utility as a quality control marker. Indeed, Bio3D grafts generated under YAP/TAZ inhibition exhibited markedly reduced CTGF production (Supplementary Fig. 2) and failed to promote periodontal tissue regeneration (Fig. 5 ), suggesting that CTGF could serve as a practical reference for establishing potency assays in future clinical development. In addition, previous studies have reported that CTGF promotes cementoblastic differentiation of periodontal ligament stem cells and contributes to cementum formation 49 , 50 . Thus, CTGF secreted by Bio3D grafts may have directly supported periodontal tissue reconstruction in addition to serving as a biomarker. Collectively, our findings indicate that CTGF production strongly reflects the periodontal regenerative potential of Bio3D grafts and may provide a simple, reliable parameter for quality control in translational and clinical applications. As described above, this study elucidated the efficacy, mode of action, safety, and potential quality control metrics of human MSC-derived Bio3D grafts fabricated with a Bio3D printer. For clinical translation, however, it is essential to address concerns regarding variability among MSC lots. Previous basic and clinical studies have consistently reported that MSC properties can vary substantially between laboratories and donors 51 , compromising reproducibility and yielding heterogeneous regenerative outcomes 52 . To evaluate whether our approach could mitigate donor-to-donor variability, we fabricated Bio3D grafts from four independent human MSC lots using the same culture and assembly process, and assessed their regenerative effects in a nude rat periodontal dehiscence defect model. Micro-CT analysis demonstrated that, at 4 weeks post-transplantation, all four donor-derived Bio3D grafts significantly promoted alveolar bone regeneration compared with untreated no-graft controls (Supplementary Fig. 3). These results suggest that the method established in this study—combining C-MSC preparation with Bio3D printing—provides robust reproducibility across different donor cell sources. Although larger sample sizes will be required to rigorously confirm this, the absence of ineffective grafts among the four lots tested suggests that Bio3D grafts may help overcome the donor-related variability and the inconsistency that have long limited conventional MSC-based transplantation approaches. This reproducibility highlights Bio3D grafts as a reliable and innovative scaffold-free strategy for periodontal tissue regeneration with strong potential for clinical translation. In conclusion, this study demonstrates that human Bio3D grafts fabricated from C-MSCs using the Bio3D printer Regenova® can achieve safe and reproducible regeneration of functional periodontal tissues in a preclinical critical-size periodontal defect model in immunodeficient rodents. The grafts not only provide structural adaptability to fit complex defects but also enhance intrinsic cellular properties through elevated YAP/TAZ activity, which is required for appropriate lineage differentiation and tissue reconstruction. Looking ahead, large-animal preclinical studies with greater clinical relevance will be essential to bridge toward human periodontitis patients, paving the way for future clinical trials and eventual clinical implementation of Bio3D graft therapy. Methods Preparation of human C-MSCs Human bone marrow MSCs purchased from LONZA (Basel, Switzerland) were cultured and expanded using xeno-free/serum-free (XF) culture medium (Prime-XV MSC XSFM MDF1; FUJIFILM Irvine Scientific, Santa Ana, CA, USA). Then, C-MSCs were generated as previously reported with minor modifications 53 . Briefly, MSCs were seeded at a high cell density of 1.0×10 5 cells/well in 48-well plates (Corning, Corning, NY, USA) and cultured in XF culture medium for 2 days. Then, confluent cells that had formed the cellular sheets with MSC-derived ECM were detached from the culture plate using a micropipette tip and transferred to a 48-well ultra-low-binding plate (Corning). After 24 h of incubation, the floating MSCs/ECM complexes rolled up to generate a round cell clump. The cell clumps so called C-MCSs were maintained in XF culture medium for 4 days (Fig. 1 A (a,b)). Generation of the Bio3D graft To fabricate the Bio3D graft, C-MSCs were robotically placed onto the needles of an array and arranged in a three-dimensional configuration according to a pre-designed 3D model using a Bio3D printer (Regenova; Cyfuse Biomedical K.K., Tokyo, Japan). Specifically, 16 C-MSCs, arranged in four columns and four rows, were maintained on the needle array in XF culture medium for 8 days. During this period, adjacent C-MSCs fused to form a single block-like construct, termed Bio3D graft (Fig. 1 A (c,d)). In addition, to evaluate the biological role of the YAP/TAZ activity induced by the Bio3D printing process, C-MSCs assembled on the needle arrays by Regenova were cultured in the presence or absence of 300 nM of YAP/TAZ inhibitor II (HY-147322, MedChemExpressm, NJ, USA) for 8 days (Fig. 5 A). Histological and Immunofluorescence Analysis of C-MSCs and Bio3D graft C-MSCs and Bio3D graft were fixed with 4% paraformaldehyde in PBS. The samples were embedded in paraffin. Eight-micrometer-thick sections were prepared. The samples were then stained with hematoxylin and eosin (HE) and observed using the Keyence BZ-X800 microscope (Keyence, Osaka, Japan). Regarding immunofluorescence analysis, the fixed samples were embedded in paraffin, and 20-µm-thick sections were used. The sections were deparaffinized, rehydrated, and incubated with L.A.B solution (Polysciences, Inc., Warrington, PA, USA) for antigen retrieval at room temperature for 10 minuntes. The sections were blocked with Blocking One Histo (Nacalai Tesque, Inc., Kyoto, Japan) at room temperature for 5 minutes and then incubated with rabbit monoclonal anti-human COL1 IgG (EPR7785; Abcam, Cambridge, MA, USA) and a rabbit anti YAP/ TAZ IgG antibody (D24E4; Cell Signaling, Beverly, MA, USA). The samples were then stained with Alexa Fluor 488 goat anti-rabbit IgG antibody (Thermo Fisher Scientific, Inc., Waltham, MA, USA) for 2 hours at room temperature. DAPI (5 µg/mL; Thermo Fisher Scientific, Inc.) was employed to counterstain nuclei. The stained samples were observed using the Keyence (Osaka, Japan) BZ-X800 microscope. To assess YAP/TAZ distribution patterns, we quantified cells in three random fields of view (n = 80 ~ 100). The patterns were classified as predominantly nuclear ( N > C) , diffuse ( N = C ), or predominantly cytoplasmic / undetectable ( N < C or undetectable). The proportion of each distribution pattern was calculated as the number of cells in that pattern divided by the total number of cells, multiplied by 100. Surgical procedures To assess the periodontal tissue regenerative property of Bio3D graft, all male F344/NJcl-rnu/rnu rats (8–9 weeks old) (Charles River Laboratories Japan, Yokohama, Japan) were used in this study after approval had been obtained from the Animal Care Committee of Hiroshima University (Protocol #A22-35). All rats were housed under specific pathogen-free (SPF) conditions at a consistent temperature of 23°C and a relative humidity of 40–60%. Animals were group-housed on a 12-hour light/dark cycle with ad libitum access to food and water. A rat mandibular periodontal dehiscence defect model was established with minor modifications to previously described protocols 18 , 23 . Briefly, rats were anaesthetized with an intraperitoneal injection of medetomidine (0.1825 mg/kg), midazolam (1 m/kg), and butorphanol tartrate (1.25 mg/kg). The skin at the surgical site was shaved and disinfected with povidone–iodine. A 1.5 cm incision along the mandible’s inferior border exposed the masseter muscle. The lower attachment of the masseteric ligament was dissected, and the flap was reflected to allow adequate access to the first molar region. The buccal bone and the first molar’s central buccal root were removed by using a rotatory instrument to expose its distal root. The periodontal ligament and cementum on the central buccal root were then carefully scraped off using hand instruments. A standardized dehiscence defect (4 mm × 3 mm × 1 mm; height × width × depth) was created (Fig. 1 D). The Bio3D graft was transplanted into the defect without any artificial scaffold, and a no-graft group served as the control (n = 3–5/each group for 2, 4, and 8 weeks observation, respectively). The masseter and skin were closed with 5 − 0 silk sutures (Mani, Tochigi, Japan). Micro-CT Analysis Rats were sacrificed at 2, 4, and 8 weeks after surgery, and the mandible region was scanned by using a CosmoScan GXⅢ (Rigaku, Tokyo, Japan) in vivo µCT with the following conditions: 100kV, 120µA ,0.025 mm / pixel and 120 seconds exposure time. Scanned data were reconstructed with CosmoScan 3D viewer 5.1.3 (Rigaku). The 3D images were aligned with the DataViewer (Bruker, Billerica, MA, USA). For bone volume analysis, the region of interest (ROI) was the buccal alveolar bone, starting from the appearance of the distal root of the first molar, which is composed of 16 two-dimensional slices (approximately 400 µm). The vertical lower limit of the ROI was set at the apical end of the distal root. Segmentation of the ROI and following bone volume measurement were performed by CT-An software (Bruker) with a threshold range of 80–255. Tissue Preparation and Histological Analysis Rats were sacrificed at 2, 4, and 8 weeks after surgery. Mandibular tissues were collected, fixed with 10% neutral buffered formalin (NBF) overnight, and decalcified with 10% Ethylenediaminetetraacetic acid (pH 7.4) for 30 days. After decalcification, the samples were dehydrated through grade ethanol, cleared with xylene, and embedded in paraffin. Semi-serial sections (8 µm) were prepared. These sections were used for HE staining and observed using light microscopy. The length of newly formed cementum in HE-stained samples was measured by using ImageJ software (National Institutes of Health). Measurements were taken along the denuded root surface of the distal root of the first molar, and the proportion of newly formed cementum length to the total denuded surface was calculated. To detect human vimentin and periostin, immunofluorescence analysis was conducted. 20-µm-thick sections were prepared. The sections were deparaffinized, rehydrated, and incubated with L.A.B. Solution (Polysciences) at room temperature for 10 minutes. Non-specific binding was blocked with Blocking One Histo (Nacalai Tesque) at room temperature for 5 minutes. These sections were immunostained with the rabbit monoclonal anti-human vimentin IgG antibody (SP20; Abcam, Cambridge, MA, USA) and mouse monoclonal anti-periostin IgG antibody (F-10; Santa Cruz Biotechnology, Dallas, TX, USA). The samples were then stained with Alexa Fluor 488 goat anti-rabbit IgG antibody (Invitrogen, Carlsbad, CA, USA) or Alexa Fluor 594 goat anti-mouse IgG antibody (Invitrogen, Carlsbad, CA, USA) for 2 hours at room temperature. Then, the nuclei were counterstained with DAPI (Invitrogen, 5 mg/mL). After washing with PBS, we detected fluorescence signals using the Keyence (Osaka, Japan) BZ-X800 microscope. To quantify host rat and donor human cells at 2, 4, and 8 weeks after surgery, vimentin-positive and vimentin-negative cells were counted in the periodontal ligament, alveolar bone, and peripheral regions of the defect. ELISA C-MSCs (1 × 10 5 cells per clump) were cultured in 250 µL of medium for 2 days, and the supernatant was collected for ELISA of CTGF using a Human CTGF ELISA Kit (ab261851, Abcam). Bio3D grafts composed of 16 C-MSC clumps were cultured in a larger volume of medium under the same conditions, and the supernatant was collected in the same manner. Because the Bio3D grafts required a greater culture volume, CTGF production was normalized to an equivalent condition of 250 µL medium per C-MSC clump to allow direct comparison with single C-MSC clumps. RNA-seq Analysis ChEA3 Total RNA was extracted using RNA-iso (Takara). The construction and sequencing of the cDNA library were carried out by the Beijing Genomics Institute using the DNBSEQ platform. Raw sequence data were filtered with SOAPnuke v2.3, and the resulting clean reads were mapped to the reference genome using HISAT2 v2.0.4. Subsequent alignment was carried out with Bowtie2 2.2.5, and gene expression levels were quantified using RSEM v1.2.8. The sequence data analysis, including differential gene expression analysis (DEGs) performed with the DESeq2 package v1.48.1, Gene Ontology (GO) enrichment analysis and heatmap, was conducted in R v4.5.1 using the R studio. A fold-change threshold of ≥ 1.5 and an adjusted P value < 0.05 were applied to identify DEGs. Upstream transcription factors associated with genes from enriched GO terms “collagen-containing extracellular matrix” and “actin filament bundle” were predicted using the web-based ChIP-X Enrichment Analysis, v3 (ChEA3) platform, which integrates ENCODE, ReMap, and several independently published CHIP-seq datasets 54 . Statistical Analysis Statistical analyses were performed using Prism software (GraphPad, La Jolla, CA, USA). A two-tailed unpaired Welch’s t -test was used to compare the means between two groups, while one-way analysis of variance (ANOVA) with Tukey’s test was used to compare multiple groups. A p -value < 0.05 was considered statistically significant. Data Availability All data are available in the main text or the supplementary materials. The raw datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request. The data of RNA-seq generating in this study has been deposited NCBI’s Gene Expression Omnibus (GEO) and can be accessed under the GEO Series Accession Number GSE309551. Declarations Acknowledgements We would like to thank the Analysis Center of Life Science, Natural Science Center for Basic Research and Development, Hiroshima University, where a part of this work was carried out. Funding This work was supported in part by the Japan Society for the Promotion of Science (JSPS) KAKENHI Grant-in-Aid for Scientific Research (no. JP24K198960A, JP24K235890A), JSPS Program for Forming Japan’s Peak Research Universities (J-PEAKS) Grant Number JPJS00420230011 and Japan Agency for Medical Research and Development (AMED) under Grant Number 25bm1223015h0003. Cyfuse Biomedical K.K. provided financial support for this work. Author Contributions H.Y., M.Y., and M.K. conceptualized and designed the overall study. H.Y., M.S., and H.S. conducted the in vivo experiments. H.Y., M.Y., M.S., H.T., Y.T., and T.M. performed the in vitro experiments. H.Y., M.Y., M.S., H.S., and T.Y. carried out data analysis. T.Y., T.A., T.S., S.H., S.M., M.I., and M.K. supervised all experiments and data analysis. H.Y., M.Y., M.S., and M.K. wrote the manuscript. All authors read and approved the manuscript. Competing Interests Harumi Takai, Yoko Torii, and Toshihiko Maekawa are employees of Cyfuse Biomedical K.K. References Pihlstrom, B. L., Michalowicz, B. S. & Johnson, N. W. Periodontal diseases. The Lancet 366 , 1809–1820 (2005). Darveau, R. P. Periodontitis: a polymicrobial disruption of host homeostasis. Nat. Rev. Microbiol. 8 , 481–490 (2010). Beukers, N. G. F. M., Van Der Heijden, G. J. M. G., Van Wijk, A. J. & Loos, B. G. Periodontitis is an independent risk indicator for atherosclerotic cardiovascular diseases among 60 174 participants in a large dental school in the Netherlands. J. Epidemiol. Community Health 71 , 37–42 (2017). Kinane, D. F., Stathopoulou, P. 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Duchamp De Lageneste, O. et al. Periosteum contains skeletal stem cells with high bone regenerative potential controlled by Periostin. Nat. Commun. 9 , 773 (2018). Yin, B. et al. Identification of Postn+ periosteal progenitor cells with bone regenerative potential. JCI Insight 9 , e182524 (2024). Chen, X. et al. Role of YAP/TAZ in bone diseases: A transductor from mechanics to biology. J. Orthop. Transl. 51 , 13–23 (2025). Ma, J., Fan, H. & Geng, H. Distinct and overlapping functions of YAP and TAZ in tooth development and periodontal homeostasis. Front. Cell Dev. Biol. 11 , 1281250 (2024). Halder, G., Dupont, S. & Piccolo, S. Transduction of mechanical and cytoskeletal cues by YAP and TAZ. Nat. Rev. Mol. Cell Biol. 13 , 591–600 (2012). Panciera, T., Azzolin, L., Cordenonsi, M. & Piccolo, S. Mechanobiology of YAP and TAZ in physiology and disease. Nat. Rev. Mol. Cell Biol. 18 , 758–770 (2017). Yuda, A. et al. Effect of CTGF/CCN2 on Osteo/Cementoblastic and Fibroblastic Differentiation of a Human Periodontal Ligament Stem/Progenitor Cell Line. J. Cell. Physiol. 230 , 150–159 (2015). Wu, Z. et al. Connective tissue growth factor promotes cementogenesis and cementum repair via Cx43/β-catenin axis. Stem Cell Res. Ther. 13 , 460 (2022). Martin, I., Galipeau, J., Kessler, C., Le Blanc, K. & Dazzi, F. Challenges for mesenchymal stromal cell therapies. Sci. Transl. Med. 11 , eaat2189 (2019). Calcat-i-Cervera, S. et al. Harmonised culture procedures minimise but do not eliminate mesenchymal stromal cell donor and tissue variability in a decentralised multicentre manufacturing approach. Stem Cell Res. Ther. 14 , 120 (2023). Kamiya, D. et al. Induction of functional xeno-free MSCs from human iPSCs via a neural crest cell lineage. Npj Regen. Med. 7 , 47 (2022). Keenan, A. B. et al. ChEA3: transcription factor enrichment analysis by orthogonal omics integration. Nucleic Acids Res. 47 , W212–W224 (2019). Additional Declarations Competing interest reported. Harumi Takai, Yoko Torii, and Toshihiko Maekawa are employees of Cyfuse Biomedical K.K. Supplementary Files SupplementaryFigure.pdf Cite Share Download PDF Status: Under Revision Version 1 posted Editorial decision: Revision requested 13 Apr, 2026 Reviews received at journal 30 Mar, 2026 Reviewers agreed at journal 23 Mar, 2026 Reviews received at journal 01 Mar, 2026 Reviewers agreed at journal 19 Feb, 2026 Reviewers invited by journal 19 Feb, 2026 Editor assigned by journal 06 Nov, 2025 Submission checks completed at journal 03 Nov, 2025 First submitted to journal 26 Oct, 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. 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11:31:24","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7953573/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7953573/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":103341116,"identity":"81b31702-2518-45ba-bbed-691c40363194","added_by":"auto","created_at":"2026-02-24 15:29:53","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":720092,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGeneration of Bio3D graft from bone marrow MSCs with xeno free conditions.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA) Schematic figure of the experimental procedure for Bio3D graft preparation. Bone marrow derived mesenchymal stem cells (MSCs) were cultured on a plate to generate a cell sheet. The cell sheets were then transferred to a low attachment plate to generate clumps of the MSC/ECM complex, C-MSCs. C-MSCs were arranged into a needle array to generate the Bio3D graft, which was then transplanted into a nude rat periodontal dehiscence defect model. Images show (a) a cell sheet, (b) C-MSCs, (c) 3D printing, and (d) a Bio3D graft. B) HE-stained images of C-MSCs and Bio3D graft and C) immunofluorescence images of human COL1 (green) with DAPI counterstaining for nuclei (blue). For both panels, upper panels show C-MSCs and lower panels show Bio3D graft. The left panels show low-magnification views (Scale bars = 1000 µm); the right panels show higher-magnification views of the boxed regions (Scale bars = 100 µm). D) Micro-CT images of a standardized dehiscence periodontal defect model, including three-dimensional reconstructed, coronal section, and transverse sections. A 4 × 3 ×1 mm periodontal defect was created by removing the mandibular alveolar bone, periodontal ligament, cementum, and dentin. E) Macroscopic images of defects without graft (no graft) and with Bio3D graft. Scale bars = 1 mm.\u003c/p\u003e","description":"","filename":"OnlineFigure.1.png","url":"https://assets-eu.researchsquare.com/files/rs-7953573/v1/1ba71daba175a95eb27fe125.png"},{"id":103506511,"identity":"e46c9cc6-cdf9-47ee-803e-22d3a46955bc","added_by":"auto","created_at":"2026-02-26 13:37:12","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":864637,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTransplantation of Bio3D graft generated in xeno-free conditions can induce rat periodontal tissue regeneration.\u003c/strong\u003e A) Animals were sacrificed at 2, 4, and 8 weeks after transplantation of the Bio3D graft, and the periodontal tissues were fixed. Representative µCT images at 2, 4, and 8 weeks after surgery are shown. Red arrows indicate a radiolucent space at 8 weeks post-surgery. B) HE-stained images of the Bio3D graft transplantation group at 2, 4, and 8 weeks after surgery, and the no graft control group at 8 weeks. The lower panels show higher-magnification views of the boxed regions. Scale bars = 500 µm. C) Quantitative analysis of the bone volume (BV) to total volume (TV) ratio in the defect regions at 2, 4, and 8 weeks after surgery. * \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 by one-tailed Welch’s \u003cem\u003et\u003c/em\u003e-test. The length of new cementum formed along the denuded root surface, shown as a percentage of the total length at 2, 4, and 8 weeks after surgery. ** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01; Welch’s \u003cem\u003et\u003c/em\u003e-test.\u003c/p\u003e","description":"","filename":"OnlineFigure.2.png","url":"https://assets-eu.researchsquare.com/files/rs-7953573/v1/ee73dce389162e9bfb079583.png"},{"id":103506006,"identity":"203bd972-ea6b-4304-b187-93c08e83717d","added_by":"auto","created_at":"2026-02-26 13:33:48","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":721221,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePeriodontal tissue regeneration induced by the Bio3D graft is facilitated by transplantation of human donor cells.\u003c/strong\u003e A) Coronal sections were prepared, and immunostained with an anti-human vimentin antibody (green). Nuclei (blue) were counterstained with DAPI. Left panels show low-magnification views (Scale bars = 500 µm), and the right panels show higher-magnification views of the periodontal ligament (upper right) and alveolar bone (lower right) (Scale bars = 50 µm). D, dentin; NB, newly formed bone. B) Immunofluorescence images of anti-human vimentin and anti-periostin. Nuclei were counterstained with DAPI. (Left panel: low-magnification, scale bar = 500 µm; right three panels: higher-magnification, scale bars = 100 µm) C) Quantitative analysis of the number of host rat cells and donor human cells at 2, 4, and 8 weeks after surgery. The periodontal ligament, alveolar bone, and peripheral region of the defect were used for counting human vimentin-positive and -negative cells. ** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01; one-way ANOVA with Tukey’s test. D) The number of host rat cells and donor human cells at 8 weeks post-surgery. Left graph: measurements in the periodontal ligament space; right graph: measurements in the alveolar bone. NS, not significant; ** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01; Welch’s \u003cem\u003et\u003c/em\u003e-test.\u003c/p\u003e","description":"","filename":"OnlineFigure.3.png","url":"https://assets-eu.researchsquare.com/files/rs-7953573/v1/498eaed7d7a1e81fd1316741.png"},{"id":103341118,"identity":"3a9c4f9c-4540-4df4-a35c-dd2e692a3fa3","added_by":"auto","created_at":"2026-02-24 15:29:53","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":323468,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFabrication of Bio3D graft by Bio3D printing enhances YAP/TAZ activity and the expression of their downstream target genes.\u003c/strong\u003e A) Differential gene expression (DEG) analysis comparing C-MSCs with the Bio3D grafts. B) Bubble charts show enrichment of cellular component categories identified by Gene Ontology analysis of genes with higher expression in the Bio3D graft. C) ChEA3 analysis listing the top four transcription factors inferred from genes enriched in the collagen-containing extracellular matrix and actin filament bundle categories. D) Heat map showing the expression intensity of YAP, TAZ, their downstream target genes and genes related to osteogenesis and periodontium. E) Immunofluorescence images of YAP/TAZ (green) and nuclei (blue) in C-MSCs and Bio3D graft. Scale bars = 20 μm. F) Quantification of YAP/TAZ localization patterns. The patterns were classified as predominantly nuclear (\u003cem\u003eN \u003c/em\u003e\u0026gt; \u003cem\u003eC),\u003c/em\u003e diffuse (\u003cem\u003eN\u003c/em\u003e = \u003cem\u003eC\u003c/em\u003e), or predominantly cytoplasmic / undetectable (\u003cem\u003eN \u003c/em\u003e\u0026lt; \u003cem\u003eC\u003c/em\u003e or undetectable). G) CTGF levels in the culture supernatant were measured by ELISA. ** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01; Welch’s \u003cem\u003et\u003c/em\u003e-test.\u003c/p\u003e","description":"","filename":"OnlineFigure.4.png","url":"https://assets-eu.researchsquare.com/files/rs-7953573/v1/a80e7667764ff8832f5447e9.png"},{"id":103341119,"identity":"7537ee04-35a5-4fb6-a899-426479c9d709","added_by":"auto","created_at":"2026-02-24 15:29:53","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":632378,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eYAP/TAZ activity induced by the Bio3D printing process is required for Bio3D graft–mediated periodontal tissue regeneration. \u003c/strong\u003eA) Schematic figure of the experimental procedure for the inhibition of YAP/TAZ activity by the specific inhibitor. B) Macroscopic images of defects with Bio3D graft under control and YAP/TAZ inhibitor II conditions. Scale bars = 1 mm. C) Representative μCT images at 4 weeks after transplantation of Bio3D grafts treated with or without YAP/TAZ inhibitor II, including three-dimensional reconstructed, coronal, and transverse sections. D) Quantitative analysis of the bone volume (BV) to the total volume (TV) ratio in the defect regions at 4 weeks after surgery. * \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05; Welch’s \u003cem\u003et\u003c/em\u003e-test. E) HE-stained images of Bio3D graft with or without YAP/TAZ inhibitor II at 4 weeks after surgery. Left panels show low-magnification views; Right panels show higher-magnification images of the boxed regions. Scale bars = 500 μm. F) The length of newly formed cementum along the denuded root surface, expressed as a percentage of the denuded surface length at 4 weeks after surgery. ** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01; Welch’s \u003cem\u003et\u003c/em\u003e-test. G) Immunofluorescence images of human vimentin (green) and nuclei counterstained with DAPI (blue). Low-magnification images show periodontal tissues from the control group (left) and from samples treated with YAP/TAZ inhibitor II (right). Higher-magnification images of the boxed regions are shown in the right panels, respectively. Scale bars = 1000 µm (low-magnification) and 100 µm (higher-magnification). H) Quantitative analysis of host rat cells and donor human cells in control and YAP/TAZ inhibitor II–treated samples at 4 weeks after surgery. Cell numbers were determined by counting vimentin-positive (human) and vimentin-negative (rat) cells in the periodontal ligament, alveolar bone, and peripheral regions of the defect. NS, not significant as determined by one-way ANOVA with Tukey’s test.\u003c/p\u003e","description":"","filename":"OnlineFigure.5.png","url":"https://assets-eu.researchsquare.com/files/rs-7953573/v1/35a6d800f3e3171015bedd5d.png"},{"id":103509791,"identity":"77a55cf0-21b8-4c6b-9bc0-01ba466580c7","added_by":"auto","created_at":"2026-02-26 14:01:15","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5398662,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7953573/v1/2b6568bb-cc50-4886-bf3d-7376a7d5506e.pdf"},{"id":103341121,"identity":"d34006fa-9ac9-47cd-a6b0-86c84ce08e5b","added_by":"auto","created_at":"2026-02-24 15:29:54","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":440950,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigure.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7953573/v1/79615dd8d3452a75614e2be0.pdf"}],"financialInterests":"Competing interest reported. Harumi Takai, Yoko Torii, and Toshihiko Maekawa are employees of Cyfuse Biomedical K.K.","formattedTitle":"Bio3D graft engineered from human mesenchymal stromal cells/extracellular complexes facilitate periodontal tissue regeneration","fulltext":[{"header":"Introduction","content":"\u003cp\u003ePeriodontitis is a chronic inflammatory disease caused by the host immune response to bacterial infection, leading to the progressive destruction of periodontal tissues, including cementum, periodontal ligament, and alveolar bone\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Without appropriate treatment, this breakdown ultimately results in tooth loss. Moreover, an increasing body of evidence suggests that periodontitis and tooth loss contribute to the exacerbation of various systemic diseases, such as dementia, pneumonia, diabetes, cardiovascular disease, and hepatitis\u003csup\u003e\u003cspan additionalcitationids=\"CR4\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Therefore, establishing a curative therapy that enables true periodontal tissue regeneration is crucial for extending healthy life expectancy in aging societies.\u003c/p\u003e \u003cp\u003eMesenchymal stromal/stem cells (MSCs), originally identified in the bone marrow, contribute to tissue homeostasis through their multipotent differentiation and self-renewal capabilities\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. Consequently, regenerative therapies using MSCs have attracted considerable medical and scientific attention over the past two decades\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. In the context of periodontal regeneration, numerous preclinical and clinical studies have explored autologous MSC transplantation combined with biomaterial scaffolds\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. While these strategies have demonstrated promising outcomes, their clinical efficacy in humans remains variable and often suboptimal\u003csup\u003e\u003cspan additionalcitationids=\"CR12\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. One critical limitation lies in the use of artificial scaffolds, which, as foreign materials, are not inherently compatible with the regeneration of authentic periodontal tissue. Successful regeneration requires that such scaffolds be gradually degraded and replaced by host-derived periodontal components\u0026mdash;a process that can delay healing and introduce risks of bacterial infection and adverse inflammatory responses.\u003c/p\u003e \u003cp\u003eTo overcome these challenges, we previously developed a scaffold-free transplantation method using clumps of MSCs and their self-produced extracellular matrix (C-MSCs). Unlike small MSC spheroids (typically\u0026thinsp;\u0026lt;\u0026thinsp;500 \u0026micro;m) that are generated predominantly through cell-cell contacts, C-MSCs are approximately 1 mm in diameter and are composed of self-produced type I collagen\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e\u0026mdash;the principal extracellular matrix component of periodontal and bone tissues\u0026mdash;and can be transplanted directly into defects without the need for artificial materials\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. C-MSCs not only eliminate the necessity for scaffold degradation but also provide an intrinsic biological microenvironment that supports cell viability, site-specific differentiation, and periodontal tissue and calvaria bone reconstruction\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e,\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. In addition, the functional properties of C-MSCs, including ECM composition, osteogenic potential, and immunomodulatory capacity, can be modulated in vitro by altering culture conditions prior to transplantation\u003csup\u003e\u003cspan additionalcitationids=\"CR20\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eTo further advance this approach toward clinical application, improvements in graft size, handling properties, and functional performance were required. In this study, we addressed these needs by employing Bio3D printing technology\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e to fabricate a novel, large-sized, scaffold-free MSC construct, which we termed the Bio3D graft. This construct was generated by skewering individual C-MSCs onto a fine needle-array using a Bio3D printer, allowing the clumps to fuse into a cohesive three-dimensional tissue-like structure. This needle-array-based assembly technique enables precise control of graft shape and size while maintaining a fully scaffold-free composition composed entirely of viable MSCs and their endogenous extracellular matrix.\u003c/p\u003e \u003cp\u003eTo establish non-clinical proof of concept (PoC) for periodontal tissue regenerative therapy using the Bio3D graft, we fabricated human Bio3D grafts by combining human bone marrow-derived MSCs with the clump culture method and Bio3D printing technology. The generated grafts were transplanted into a nude rat periodontal dehiscence defect model, which represents one of the largest standardized-size critical defects in immunodeficient animals. We further analyzed the unique biological characteristics of the Bio3D grafts and investigated the underlying mechanisms of tissue regeneration. Compared to conventional C-MSCs, which are limited to small spherical clumps (~\u0026thinsp;1 mm), the Bio3D graft exhibited a structurally robust, large-scale architecture that allowed easy transplantation into large defects without the need for artificial scaffolds. The graft consistently induced reliable periodontal tissue regeneration. Notably, the Bio3D grafts demonstrated enhanced YAP/TAZ signaling activity, which facilitated appropriate differentiation of the transplanted human cells and led to successful reconstruction of periodontal tissues. Taken together, the present study highlights the potential of the Bio3D graft as an innovative, scaffold-free regenerative approach in which MSCs self-organize into large, clinically applicable implants capable of reconstructing complex periodontal tissues.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eGeneration of human Bio3D grafts tailored to the nude rat periodontal dehiscence defect model\u003c/h2\u003e \u003cp\u003eWe first generated clumps of mesenchymal stromal cells and extracellular matrix (C-MSCs) approximately 1.2 mm in diameter from human bone marrow-derived MSCs, as described in the Materials and Methods (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA (a,b)). Histological and immunofluorescence analyses revealed that C-MSCs consisted of viable cells embedded within a self-produced type I collagen (COL1) matrix (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB, \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC, upper panels). Previously, it was demonstrated that transplantation of multiple C-MSCs without artificial scaffolds into a 2 mm \u0026times; 3 mm \u0026times; 1 mm rat periodontal fenestration defect\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e\u0026mdash;an accessible defect model in which the cervical area of alveolar bone is preserved\u0026mdash;successfully induced periodontal tissue regeneration\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. To determine whether scaffold-free C-MSCs transplantation could also be effective in a more challenging setting, we transplanted twelve human C-MSCs into a 4 mm \u0026times; 3 mm \u0026times; 1 mm nude rat periodontal dehiscence defect model developed as a modification of the fenestration defect model, in which the cervical bone is completely removed, creating a standardized-size critical defect (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). This dehiscence model mimics severe periodontitis with direct communication between the defect and the oral cavity. However, C-MSCs transplantation failed to induce effective periodontal tissue regeneration compared with the no-graft control (Supplementary Fig.\u0026nbsp;1), possibly due to insufficient engraftment of transplanted C-MSCs in the absence of cervical bone support and the inability of small clumps to exert adequate regenerative function within such a large defect.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe therefore sought to fabricate a Bio3D graft precisely matching the geometry of the nude rat dehiscence defect, enabling stable engraftment and more effectively exerting cellular functions. To this end, C-MSCs were assembled using a Bio3D printer by stacking them on a fine needle-array in a 4 \u0026times; 4 arrangement, allowing them to fuse into a single construct (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA (c)). After eight days of culture, this process yielded a structurally stable approximately 3\u0026ndash;4 mm square tissue construct, hereafter referred to as the Bio3D graft (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA (d)). Histological examination confirmed that, similar to the original C-MSCs, the Bio3D graft was composed of viable cells embedded in a COL1 matrix. As anticipated, the fabricated Bio3D graft could be easily placed directly into the 4 mm \u0026times; 3 mm \u0026times; 1 mm dehiscence defect without the need for artificial materials, achieving full coverage of the defect site (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eHuman Bio3D graft induces successful periodontal tissue regeneration in a nude rat dehiscence defect model\u003c/h3\u003e\n\u003cp\u003eSince the human Bio3D graft could be successfully placed into the standardized-size nude rat periodontal dehiscence defect (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), we next monitored its effect on alveolar bone regeneration using micro-computed tomography (micro-CT). In the no-graft group, minimal bone formation was observed up to 4 weeks post-surgery, and even at 8 weeks the defect remained largely unhealed, with persistent root exposure (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). In contrast, in the Bio3D graft group, early bone formation was evident early as 2 weeks post-transplantation. By 4 weeks, regenerated bone fully covered the previously exposed root surfaces, and by 8 weeks the defect was completely filled with mature bone (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC left graph). Notably, sagittal and horizontal micro-CT images at 8 weeks revealed a radiolucent space between the root surface and the newly formed bone (red arrows in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA), suggesting preservation of a functional periodontal ligament space without ankylosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, lower right panels).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo determine whether the Bio3D graft could induce appropriate regeneration of all periodontal components\u0026mdash;cementum, periodontal ligament, and alveolar bone\u0026mdash;we performed histological analyses using HE staining. Consistent with the CT findings, the no-graft group at 8 weeks exhibited limited periodontal tissue, with the defect site largely occupied by granulation tissue-like soft connective tissue (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). In contrast, by 4 weeks post-transplantation, the Bio3D graft group demonstrated periodontal tissue regeneration covering the root surface. At 8 weeks, high-magnification views clearly showed a periodontal ligament structure connecting the newly formed alveolar bone and newly deposited cementum along the root surface, particularly in the coronal region near the top of the defect. For quantitative assessment of functional periodontal regeneration, we measured the length of newly formed cementum. The Bio3D graft group showed a significant increase in cementum formation compared with the no-graft group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC right graph). Collectively, these results indicate that the human Bio3D graft effectively promotes regeneration of functional periodontal tissues in a critical-size nude rat dehiscence defect model.\u003c/p\u003e \u003cp\u003e \u003cb\u003eTransplanted human donor cells contribute to periodontal tissue regeneration induced by the Bio3D graft.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo investigate the role of transplanted human cells in Bio3D graft\u0026ndash;induced periodontal regeneration, we performed immunofluorescence staining with an anti-human vimentin antibody to distinguish between donor human cells and host rat cells. At 2 weeks post-surgery, abundant human vimentin\u0026ndash;positive cells descended from the Bio3D graft were detected within the defect area, including in regions corresponding to newly forming alveolar bone and the periodontal ligament space at the bottom of the defect (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA (a,b)). Notably, human cells were also observed in regenerated periodontal tissues\u0026mdash;comprising cementum, ligament space, and alveolar bone\u0026mdash;even at later time points of 4 and 8 weeks post-transplantation (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA (c-f)). At 8 weeks, human cells persisted not only within the regenerated periodontal ligament space but also along the periosteal surface of the matured alveolar bone. To assess whether the donor human cells within regenerated periodontal tissue had undergone appropriate lineage differentiation, we conducted immunostaining for periostin (POSTN), a marker of periodontal ligament fibroblasts\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. Multiplex staining demonstrated that many human vimentin\u0026ndash;positive cells located in the regenerated ligament space co-expressed periostin (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). In agreement with the known expression of periostin by periosteal cells\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e, human cells at the periosteal surface of the regenerated alveolar bone also co-expressed periostin, appearing as yellow signals in merged images (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe next quantified the number of donor-derived human cells in the regenerated tissues over time. The number of human cells decreased progressively from 2 to 8 weeks post-transplantation (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). By 8 weeks, within the regenerated ligament space, the number of human cells and host rat cells were comparable; however, in the regenerated alveolar bone, host cells were significantly more abundant than human cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). These observations suggest that Bio3D graft\u0026ndash;derived human MSCs initially differentiate into periodontal component cells and contribute directly to tissue reconstruction, and that over time, these regenerated tissues may undergo normal physiological turnover, gradually becoming populated predominantly by host-derived cells while maintaining a functional and stable periodontal architecture.\u003c/p\u003e\n\u003ch3\u003eFabrication of Bio3D grafts using Bio3D printing enhances YAP/TAZ activity\u003c/h3\u003e\n\u003cp\u003eFor clinical translation of Bio3D grafts, it is essential to understand their cellular characteristics precisely. We therefore performed bulk RNA sequencing to compare the transcriptomic profiles of the Bio3D grafts with those of the original C-MSCs used as the starting material. Differential expression analysis identified 1,245 genes that were upregulated more than 1.5-fold in the Bio3D grafts relative to C-MSCs, whereas 614 genes were downregulated (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Gene Ontology enrichment analysis of the upregulated genes revealed significant enrichment for terms related to collagen-containing extracellular matrix and actin filament bundle (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). To predict transcriptional factors potentially responsible for these changes, we queried the ChEA3 database. Notably, among the top four ranked transcriptional regulators for each term, five genes were known targets or interacting partners of the transcriptional co-activators YAP/TAZ (highlighted in red in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC)\u003csup\u003e\u003cspan additionalcitationids=\"CR26 CR27 CR28\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIt is well accepted that YAP/TAZ are key mechanotransducers in MSCs to control cell fate\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. More specifically, increased YAP/TAZ activity promotes MSCs differentiation toward osteogenic and periodontal tissue\u0026ndash;forming lineages\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e,\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. We therefore examined the expression levels of YAP/TAZ themselves, their downstream target genes, and genes associated with osteogenesis and periodontal tissue formation. As anticipated, these genes were markedly upregulated in Bio3D grafts compared with C-MSCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). The mechanotransducer YAP/TAZ shuttle between the cytoplasm and nucleus, with nuclear localization indicative of active signaling\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. Thus, to validate the RNA-seq findings at the protein level, we performed immunofluorescence staining for YAP/TAZ. In C-MSCs, YAP/TAZ predominantly localized in the cytoplasm, consistent with low activity. By contrast, a larger proportion of cells in the Bio3D grafts exhibited nuclear localization of YAP/TAZ, indicating enhanced pathway activation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE,F). Besides, the levels of CTGF production, a well-established downstream target of YAP/TAZ signaling, was significantly increased in Bio3D grafts compared to C-MSCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG). Collectively, these results demonstrate that Bio3D grafts, fabricated from C-MSCs by Bio3D printing system, not only increase construct size but also enhance YAP/TAZ activity, a mechanotransduction pathway known to facilitate osteogenic and periodontal tissue\u0026ndash;related differentiation.\u003c/p\u003e \u003cp\u003e \u003cb\u003eYAP/TAZ activity induced by the Bio3D printing process is required for Bio3D graft\u0026ndash;mediated periodontal tissue regeneration\u003c/b\u003e \u003c/p\u003e \u003cp\u003eFinally, to examine whether the YAP/TAZ activity induced by the Bio3D printing process contributes to the periodontal regenerative capacity of Bio3D grafts, we conducted an inhibition experiment. Briefly, C-MSCs were assembled on the needle array of the Bio3D printer to fabricate Bio3D grafts in the presence or absence of YAP/TAZ inhibitor II (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Treatment with the inhibitor did not cause any observable difference in graft size (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB) or degradation of extracellular matrix proteins (Supplementary Fig.\u0026nbsp;2A); however, it clearly suppressed YAP/TAZ activity, as evidenced by cytoplasmic translocation and degradation of YAP/TAZ (Supplementary Fig.\u0026nbsp;2B) and reduction in CTGF (Supplementary Fig.\u0026nbsp;2C). These results confirmed that Bio3D grafts generated with YAP/TAZ-specific inhibitor exhibited diminished YAP/TAZ activity.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe inhibitor-treated Bio3D grafts were then transplanted into a nude rat periodontal dehiscence defect model. Notably, micro-CT analysis at 4 weeks post-transplantation demonstrated that defects receiving inhibitor-treated grafts showed no appreciable alveolar bone regeneration (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC, D). In agreement with this finding, histological analysis revealed that inhibitor-treated grafts failed to reconstruct the periodontium\u0026mdash;comprising cementum, periodontal ligament, and alveolar bone\u0026mdash;and instead formed only fibrous connective tissue (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE, F). To assess the persistence of donor human cells after transplantation, immunofluorescence staining for human vimentin was performed. Importantly, the number of vimentin-positive human cells engrafted within the fibrous tissue of defects receiving inhibitor-treated grafts was comparable to that in defects receiving untreated grafts (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG, H). Collectively, these findings indicate that although human cells from inhibitor-treated Bio3D grafts can engraft within the defect, suppression of YAP/TAZ activity may prevent appropriate lineage differentiation and subsequent reconstruction of periodontal tissues. Thus, YAP/TAZ activity constitutes an intrinsic cellular property of Bio3D grafts that is essential for their periodontal regenerative function.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eWe previously developed a scaffold-free cell transplantation strategy using clumps of MSCs and their extracellular matrix proteins (C-MSCs), approximately 1 mm in diameter, which enabled bone and periodontal tissue regeneration without artificial materials\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. In the present study, we advanced this approach by employing the Bio3D printer Regenova\u0026reg; to assemble C-MSCs on a needle array and fabricate larger human Bio3D grafts with enhanced regenerative capacity. Importantly, the Bio3D grafts were not only increased in size but also exhibited elevated YAP/TAZ activity, which proved to be functionally required for their superior periodontal regenerative effects. Thus, fabrication of Bio3D grafts by Bio3D printing improves not only the handling and structural adaptability of the grafts to fit complex periodontal defects, but also enhances their intrinsic cellular properties. Collectively, these findings highlight human Bio3D grafts as an innovative scaffold-free cell therapy that combines operability with improved functionality for periodontal tissue regeneration.\u003c/p\u003e \u003cp\u003ePrevious studies have suggested that paracrine effects, rather than direct differentiation, are primarily responsible for periodontal tissue regeneration induced by grafted MSCs\u003csup\u003e\u003cspan additionalcitationids=\"CR35\" citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. In contrast, our findings indicate that, following Bio3D graft transplantation, donor human cells themselves participated in periodontal tissue reconstruction (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Specifically, cementum, periodontal ligament, and alveolar bone were regenerated through appropriate lineage differentiation of the transplanted human MSCs. This direct contribution may be explained by the unique scaffold-free architecture of the Bio3D graft. Because the graft is composed of MSCs embedded within their self-produced ECM proteins, particularly COL1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), it could provide a biologically native microenvironment at the defect site without introducing artificial scaffolds that could act as foreign bodies. Compared with conventional artificial scaffold-based MSC transplantation, this environment likely facilitated the intrinsic multipotency of MSCs, thereby promoting site-specific differentiation and tissue reconstruction by the transplanted cells themselves. In particular, as COL1 is the predominant ECM protein in bone and periodontal ligament\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e,\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e, Bio3D grafts enriched in COL1 may provide a favorable cellular microenvironment for grafted MSCs to differentiate into cementoblasts, periodontal ligament fibroblasts, and osteoblasts. Although further investigation is needed to identify the specific environmental cues that direct MSCs toward these periodontal lineages, our findings highlight that Bio3D graft\u0026ndash;mediated regeneration involves not only paracrine signaling but also a substantive contribution from the intrinsic multipotency of MSCs. This perspective revisits a classical concept of MSC-based regenerative therapy\u0026mdash;tissue reconstruction through differentiation of transplanted cells\u0026mdash;which has often been overshadowed by the paracrine paradigm in recent years\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e,\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn this study, the number of transplanted human MSCs within the defect gradually decreased over time, and importantly, no evidence of bone overgrowth, ankylosis, or ectopic tissue formation was observed at 8 weeks after surgery. These findings may support the safety of periodontal regenerative therapy using human Bio3D grafts and represent a favorable outcome from a translational perspective. At the same time, it is noteworthy that at 8 weeks post-transplantation, numerous donor-derived human cells expressing POSTN persisted within the newly formed periodontal ligament space and along the periosteal surface of alveolar bone. POSTN is highly enriched in the periodontal ligament and is indispensable for maintaining the periodontal homeostasis\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e,\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. In addition, POSTN-positive stromal stem/progenitor cells in the periosteum contribute to bone homeostasis and regeneration\u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e,\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. Thus, although the majority of transplanted cells likely disappeared after participating in tissue reconstruction through normal turnover, those that reached the stem cell niches of the periodontal ligament and periosteum may have persisted and contributed to long-term maintenance of tissue integrity. Taken together, these findings suggest that Bio3D graft transplantation can achieve safe and reliable functional regeneration of periodontal tissues without undesirable outcomes such as ectopic tissue formation or tumorigenesis, while supporting long-term maintenance of tissue homeostasis.\u003c/p\u003e \u003cp\u003eHuman Bio3D grafts fabricated from C-MSCs using the Bio3D printer Regenova\u0026reg; exhibited markedly elevated YAP/TAZ activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). In MSCs, increased YAP/TAZ signaling is well known to promote lineage commitment toward osteogenic\u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e and periodontal tissue\u0026ndash;forming cells\u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e. Thus, the reconstruction of cementum, periodontal ligament, and alveolar bone by transplanted human MSCs observed in this study is most likely attributable to enhanced YAP/TAZ activity. This interpretation is strongly supported by the finding that Bio3D grafts generated in the presence of a YAP/TAZ inhibitor retained engrafted human cells within the defect but failed to induce periodontal tissue reconstruction (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e), indicating that suppression of YAP/TAZ activity impaired appropriate differentiation. The precise molecular mechanisms responsible for the elevated YAP/TAZ activity in Bio3D grafts were not clarified in this study. However, YAP/TAZ are established mechanotransducers that convert substrate stiffness and physical forces into biochemical signals\u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e. In particular, abundant ECM deposition, increased matrix rigidity, or mechanical loading are well known to activate YAP/TAZ and direct MSCs toward osteogenic differentiation\u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e. It is therefore plausible that the Bio3D fabrication process\u0026mdash;including the stacking of C-MSCs on the needle array, the sensing of needle stiffness and the presence of abundant COL1 matrix\u0026mdash;collectively provided mechanical cues that enhanced YAP/TAZ activity.\u003c/p\u003e \u003cp\u003eBecause YAP/TAZ are transcriptional co-activators that shuttle between the cytoplasm and nucleus, their activity is often assessed indirectly by measuring the expression of downstream target genes. Among these, connective tissue growth factor (CTGF/CCN2) is a particularly reliable surrogate marker, as its expression closely reflects YAP/TAZ activity\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e,\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. Consistent with this, our study demonstrated that CTGF levels in the culture supernatant provided a convenient readout of YAP/TAZ activity in Bio3D grafts. Importantly, from a translational perspective, CTGF production not only reflects YAP/TAZ signaling but also correlates with the regenerative potency of Bio3D grafts, highlighting its potential utility as a quality control marker. Indeed, Bio3D grafts generated under YAP/TAZ inhibition exhibited markedly reduced CTGF production (Supplementary Fig.\u0026nbsp;2) and failed to promote periodontal tissue regeneration (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e), suggesting that CTGF could serve as a practical reference for establishing potency assays in future clinical development. In addition, previous studies have reported that CTGF promotes cementoblastic differentiation of periodontal ligament stem cells and contributes to cementum formation\u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e,\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e. Thus, CTGF secreted by Bio3D grafts may have directly supported periodontal tissue reconstruction in addition to serving as a biomarker. Collectively, our findings indicate that CTGF production strongly reflects the periodontal regenerative potential of Bio3D grafts and may provide a simple, reliable parameter for quality control in translational and clinical applications.\u003c/p\u003e \u003cp\u003eAs described above, this study elucidated the efficacy, mode of action, safety, and potential quality control metrics of human MSC-derived Bio3D grafts fabricated with a Bio3D printer. For clinical translation, however, it is essential to address concerns regarding variability among MSC lots. Previous basic and clinical studies have consistently reported that MSC properties can vary substantially between laboratories and donors\u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e, compromising reproducibility and yielding heterogeneous regenerative outcomes\u003csup\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e. To evaluate whether our approach could mitigate donor-to-donor variability, we fabricated Bio3D grafts from four independent human MSC lots using the same culture and assembly process, and assessed their regenerative effects in a nude rat periodontal dehiscence defect model. Micro-CT analysis demonstrated that, at 4 weeks post-transplantation, all four donor-derived Bio3D grafts significantly promoted alveolar bone regeneration compared with untreated no-graft controls (Supplementary Fig.\u0026nbsp;3). These results suggest that the method established in this study\u0026mdash;combining C-MSC preparation with Bio3D printing\u0026mdash;provides robust reproducibility across different donor cell sources. Although larger sample sizes will be required to rigorously confirm this, the absence of ineffective grafts among the four lots tested suggests that Bio3D grafts may help overcome the donor-related variability and the inconsistency that have long limited conventional MSC-based transplantation approaches. This reproducibility highlights Bio3D grafts as a reliable and innovative scaffold-free strategy for periodontal tissue regeneration with strong potential for clinical translation.\u003c/p\u003e \u003cp\u003eIn conclusion, this study demonstrates that human Bio3D grafts fabricated from C-MSCs using the Bio3D printer Regenova\u0026reg; can achieve safe and reproducible regeneration of functional periodontal tissues in a preclinical critical-size periodontal defect model in immunodeficient rodents. The grafts not only provide structural adaptability to fit complex defects but also enhance intrinsic cellular properties through elevated YAP/TAZ activity, which is required for appropriate lineage differentiation and tissue reconstruction. Looking ahead, large-animal preclinical studies with greater clinical relevance will be essential to bridge toward human periodontitis patients, paving the way for future clinical trials and eventual clinical implementation of Bio3D graft therapy.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003ePreparation of human C-MSCs\u003c/h2\u003e \u003cp\u003eHuman bone marrow MSCs purchased from LONZA (Basel, Switzerland) were cultured and expanded using xeno-free/serum-free (XF) culture medium (Prime-XV MSC XSFM MDF1; FUJIFILM Irvine Scientific, Santa Ana, CA, USA). Then, C-MSCs were generated as previously reported with minor modifications\u003csup\u003e\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e. Briefly, MSCs were seeded at a high cell density of 1.0\u0026times;10\u003csup\u003e5\u003c/sup\u003e cells/well in 48-well plates (Corning, Corning, NY, USA) and cultured in XF culture medium for 2 days. Then, confluent cells that had formed the cellular sheets with MSC-derived ECM were detached from the culture plate using a micropipette tip and transferred to a 48-well ultra-low-binding plate (Corning). After 24 h of incubation, the floating MSCs/ECM complexes rolled up to generate a round cell clump. The cell clumps so called C-MCSs were maintained in XF culture medium for 4 days (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA (a,b)).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eGeneration of the Bio3D graft\u003c/h3\u003e\n\u003cp\u003eTo fabricate the Bio3D graft, C-MSCs were robotically placed onto the needles of an array and arranged in a three-dimensional configuration according to a pre-designed 3D model using a Bio3D printer (Regenova; Cyfuse Biomedical K.K., Tokyo, Japan). Specifically, 16 C-MSCs, arranged in four columns and four rows, were maintained on the needle array in XF culture medium for 8 days. During this period, adjacent C-MSCs fused to form a single block-like construct, termed Bio3D graft (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA (c,d)). In addition, to evaluate the biological role of the YAP/TAZ activity induced by the Bio3D printing process, C-MSCs assembled on the needle arrays by Regenova were cultured in the presence or absence of 300 nM of YAP/TAZ inhibitor II (HY-147322, MedChemExpressm, NJ, USA) for 8 days (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA).\u003c/p\u003e\n\u003ch3\u003eHistological and Immunofluorescence Analysis of C-MSCs and Bio3D graft\u003c/h3\u003e\n\u003cp\u003eC-MSCs and Bio3D graft were fixed with 4% paraformaldehyde in PBS. The samples were embedded in paraffin. Eight-micrometer-thick sections were prepared. The samples were then stained with hematoxylin and eosin (HE) and observed using the Keyence BZ-X800 microscope (Keyence, Osaka, Japan). Regarding immunofluorescence analysis, the fixed samples were embedded in paraffin, and 20-\u0026micro;m-thick sections were used. The sections were deparaffinized, rehydrated, and incubated with L.A.B solution (Polysciences, Inc., Warrington, PA, USA) for antigen retrieval at room temperature for 10 minuntes. The sections were blocked with Blocking One Histo (Nacalai Tesque, Inc., Kyoto, Japan) at room temperature for 5 minutes and then incubated with rabbit monoclonal anti-human COL1 IgG (EPR7785; Abcam, Cambridge, MA, USA) and a rabbit anti YAP/ TAZ IgG antibody (D24E4; Cell Signaling, Beverly, MA, USA). The samples were then stained with Alexa Fluor 488 goat anti-rabbit IgG antibody (Thermo Fisher Scientific, Inc., Waltham, MA, USA) for 2 hours at room temperature. DAPI (5 \u0026micro;g/mL; Thermo Fisher Scientific, Inc.) was employed to counterstain nuclei. The stained samples were observed using the Keyence (Osaka, Japan) BZ-X800 microscope. To assess YAP/TAZ distribution patterns, we quantified cells in three random fields of view (n\u0026thinsp;=\u0026thinsp;80\u0026thinsp;~\u0026thinsp;100). The patterns were classified as predominantly nuclear (\u003cem\u003eN\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;\u003cem\u003eC)\u003c/em\u003e, diffuse (\u003cem\u003eN\u003c/em\u003e\u0026thinsp;=\u0026thinsp;\u003cem\u003eC\u003c/em\u003e), or predominantly cytoplasmic / undetectable (\u003cem\u003eN\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;\u003cem\u003eC\u003c/em\u003e or undetectable). The proportion of each distribution pattern was calculated as the number of cells in that pattern divided by the total number of cells, multiplied by 100.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eSurgical procedures\u003c/h2\u003e \u003cp\u003e To assess the periodontal tissue regenerative property of Bio3D graft, all male F344/NJcl-rnu/rnu rats (8\u0026ndash;9 weeks old) (Charles River Laboratories Japan, Yokohama, Japan) were used in this study after approval had been obtained from the Animal Care Committee of Hiroshima University (Protocol #A22-35). All rats were housed under specific pathogen-free (SPF) conditions at a consistent temperature of 23\u0026deg;C and a relative humidity of 40\u0026ndash;60%. Animals were group-housed on a 12-hour light/dark cycle with ad libitum access to food and water. A rat mandibular periodontal dehiscence defect model was established with minor modifications to previously described protocols\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. Briefly, rats were anaesthetized with an intraperitoneal injection of medetomidine (0.1825 mg/kg), midazolam (1 m/kg), and butorphanol tartrate (1.25 mg/kg). The skin at the surgical site was shaved and disinfected with povidone\u0026ndash;iodine. A 1.5 cm incision along the mandible\u0026rsquo;s inferior border exposed the masseter muscle. The lower attachment of the masseteric ligament was dissected, and the flap was reflected to allow adequate access to the first molar region. The buccal bone and the first molar\u0026rsquo;s central buccal root were removed by using a rotatory instrument to expose its distal root. The periodontal ligament and cementum on the central buccal root were then carefully scraped off using hand instruments. A standardized dehiscence defect (4 mm \u0026times; 3 mm \u0026times; 1 mm; height \u0026times; width \u0026times; depth) was created (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). The Bio3D graft was transplanted into the defect without any artificial scaffold, and a no-graft group served as the control (n\u0026thinsp;=\u0026thinsp;3\u0026ndash;5/each group for 2, 4, and 8 weeks observation, respectively). The masseter and skin were closed with 5\u0026thinsp;\u0026minus;\u0026thinsp;0 silk sutures (Mani, Tochigi, Japan).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eMicro-CT Analysis\u003c/h2\u003e \u003cp\u003eRats were sacrificed at 2, 4, and 8 weeks after surgery, and the mandible region was scanned by using a CosmoScan GXⅢ (Rigaku, Tokyo, Japan) in vivo \u0026micro;CT with the following conditions: 100kV, 120\u0026micro;A ,0.025 mm / pixel and 120 seconds exposure time. Scanned data were reconstructed with CosmoScan 3D viewer 5.1.3 (Rigaku). The 3D images were aligned with the DataViewer (Bruker, Billerica, MA, USA). For bone volume analysis, the region of interest (ROI) was the buccal alveolar bone, starting from the appearance of the distal root of the first molar, which is composed of 16 two-dimensional slices (approximately 400 \u0026micro;m). The vertical lower limit of the ROI was set at the apical end of the distal root. Segmentation of the ROI and following bone volume measurement were performed by CT-An software (Bruker) with a threshold range of 80\u0026ndash;255.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eTissue Preparation and Histological Analysis\u003c/h2\u003e \u003cp\u003eRats were sacrificed at 2, 4, and 8 weeks after surgery. Mandibular tissues were collected, fixed with 10% neutral buffered formalin (NBF) overnight, and decalcified with 10% Ethylenediaminetetraacetic acid (pH 7.4) for 30 days. After decalcification, the samples were dehydrated through grade ethanol, cleared with xylene, and embedded in paraffin. Semi-serial sections (8 \u0026micro;m) were prepared. These sections were used for HE staining and observed using light microscopy. The length of newly formed cementum in HE-stained samples was measured by using ImageJ software (National Institutes of Health). Measurements were taken along the denuded root surface of the distal root of the first molar, and the proportion of newly formed cementum length to the total denuded surface was calculated. To detect human vimentin and periostin, immunofluorescence analysis was conducted. 20-\u0026micro;m-thick sections were prepared. The sections were deparaffinized, rehydrated, and incubated with L.A.B. Solution (Polysciences) at room temperature for 10 minutes. Non-specific binding was blocked with Blocking One Histo (Nacalai Tesque) at room temperature for 5 minutes. These sections were immunostained with the rabbit monoclonal anti-human vimentin IgG antibody (SP20; Abcam, Cambridge, MA, USA) and mouse monoclonal anti-periostin IgG antibody (F-10; Santa Cruz Biotechnology, Dallas, TX, USA). The samples were then stained with Alexa Fluor 488 goat anti-rabbit IgG antibody (Invitrogen, Carlsbad, CA, USA) or Alexa Fluor 594 goat anti-mouse IgG antibody (Invitrogen, Carlsbad, CA, USA) for 2 hours at room temperature. Then, the nuclei were counterstained with DAPI (Invitrogen, 5 mg/mL). After washing with PBS, we detected fluorescence signals using the Keyence (Osaka, Japan) BZ-X800 microscope. To quantify host rat and donor human cells at 2, 4, and 8 weeks after surgery, vimentin-positive and vimentin-negative cells were counted in the periodontal ligament, alveolar bone, and peripheral regions of the defect.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eELISA\u003c/h2\u003e \u003cp\u003eC-MSCs (1 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells per clump) were cultured in 250 \u0026micro;L of medium for 2 days, and the supernatant was collected for ELISA of CTGF using a Human CTGF ELISA Kit (ab261851, Abcam). Bio3D grafts composed of 16 C-MSC clumps were cultured in a larger volume of medium under the same conditions, and the supernatant was collected in the same manner. Because the Bio3D grafts required a greater culture volume, CTGF production was normalized to an equivalent condition of 250 \u0026micro;L medium per C-MSC clump to allow direct comparison with single C-MSC clumps.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eRNA-seq Analysis ChEA3\u003c/h2\u003e \u003cp\u003eTotal RNA was extracted using RNA-iso (Takara). The construction and sequencing of the cDNA library were carried out by the Beijing Genomics Institute using the DNBSEQ platform. Raw sequence data were filtered with SOAPnuke v2.3, and the resulting clean reads were mapped to the reference genome using HISAT2 v2.0.4. Subsequent alignment was carried out with Bowtie2 2.2.5, and gene expression levels were quantified using RSEM v1.2.8. The sequence data analysis, including differential gene expression analysis (DEGs) performed with the DESeq2 package v1.48.1, Gene Ontology (GO) enrichment analysis and heatmap, was conducted in R v4.5.1 using the R studio. A fold-change threshold of \u0026ge;\u0026thinsp;1.5 and an adjusted P value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 were applied to identify DEGs. Upstream transcription factors associated with genes from enriched GO terms \u0026ldquo;collagen-containing extracellular matrix\u0026rdquo; and \u0026ldquo;actin filament bundle\u0026rdquo; were predicted using the web-based ChIP-X Enrichment Analysis, v3 (ChEA3) platform, which integrates ENCODE, ReMap, and several independently published CHIP-seq datasets\u003csup\u003e\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eStatistical analyses were performed using Prism software (GraphPad, La Jolla, CA, USA). A two-tailed unpaired Welch\u0026rsquo;s \u003cem\u003et\u003c/em\u003e-test was used to compare the means between two groups, while one-way analysis of variance (ANOVA) with Tukey\u0026rsquo;s test was used to compare multiple groups. A \u003cem\u003ep\u003c/em\u003e-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eData Availability\u003c/h2\u003e \u003cp\u003eAll data are available in the main text or the supplementary materials. The raw datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request. The data of RNA-seq generating in this study has been deposited NCBI\u0026rsquo;s Gene Expression Omnibus (GEO) and can be accessed under the GEO Series Accession Number \u003cb\u003eGSE309551.\u003c/b\u003e\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe would like to thank the Analysis Center of Life Science, Natural Science Center for Basic Research and Development, Hiroshima University, where a part of this work was carried out.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported in part by the Japan Society for the Promotion of Science (JSPS) KAKENHI Grant-in-Aid for Scientific Research (no. JP24K198960A, JP24K235890A), JSPS Program for Forming Japan’s Peak Research Universities (J-PEAKS) Grant Number JPJS00420230011 and Japan Agency for Medical Research and Development (AMED) under Grant Number 25bm1223015h0003. Cyfuse Biomedical K.K. provided financial support for this work.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eH.Y., M.Y., and M.K. conceptualized and designed the overall study. H.Y., M.S., and H.S. conducted the in vivo experiments. H.Y., M.Y., M.S., H.T., Y.T., and T.M. performed the in vitro experiments. H.Y., M.Y., M.S., H.S., and T.Y. carried out data analysis. T.Y., T.A., T.S., S.H., S.M., M.I., and M.K. supervised all experiments and data analysis. H.Y., M.Y., M.S., and M.K. wrote the manuscript. All authors read and approved the manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHarumi Takai, Yoko Torii, and Toshihiko Maekawa are employees of Cyfuse Biomedical K.K.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003ePihlstrom, B. L., Michalowicz, B. S. \u0026amp; Johnson, N. W. Periodontal diseases. \u003cem\u003eThe Lancet\u003c/em\u003e \u003cstrong\u003e366\u003c/strong\u003e, 1809\u0026ndash;1820 (2005).\u003c/li\u003e\n \u003cli\u003eDarveau, R. P. Periodontitis: a polymicrobial disruption of host homeostasis. \u003cem\u003eNat. Rev. Microbiol.\u003c/em\u003e \u003cstrong\u003e8\u003c/strong\u003e, 481\u0026ndash;490 (2010).\u003c/li\u003e\n \u003cli\u003eBeukers, N. G. F. M., Van Der Heijden, G. J. M. G., Van Wijk, A. J. \u0026amp; Loos, B. G. 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B. \u003cem\u003eet al.\u003c/em\u003e ChEA3: transcription factor enrichment analysis by orthogonal omics integration. \u003cem\u003eNucleic Acids Res.\u003c/em\u003e \u003cstrong\u003e47\u003c/strong\u003e, W212\u0026ndash;W224 (2019).\u003cstrong\u003e\u003c/strong\u003e\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"npj-regenerative-medicine","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"npjregenmed","sideBox":"Learn more about [npj Regenerative Medicine](http://www.nature.com/npjregenmed/)","snPcode":"41536","submissionUrl":"https://mts-npjregenmed.nature.com/cgi-bin/main.plex","title":"npj Regenerative Medicine","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"NPJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-7953573/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7953573/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003ePeriodontitis, a chronic inflammatory disease leading to irreversible loss of tooth-supporting tissues, remains a major unmet clinical challenge despite advances in biomaterial-based cell therapies. Scaffold-free regenerative strategies that fully rely on cellular and extracellular matrix (ECM) components may overcome the limitations of artificial scaffolds. Here, we developed a human Bio3D graft fabricated from clumps of mesenchymal stromal cells and their self-produced ECM (C-MSCs) using a Bio3D printer. When transplanted into a critical-size periodontal defect in immunodeficient rats, the Bio3D graft achieved safe and reproducible regeneration of cementum, periodontal ligament, and alveolar bone without ectopic or excessive bone formation. Transcriptomic and histological analyses revealed enhanced YAP/TAZ signaling in the Bio3D graft compared with C-MSCs, and functional inhibition of YAP/TAZ abolished periodontal reconstruction, demonstrating its essential role. These findings establish a foundation for next-generation, scaffold-free cell therapies capable of reconstructing complex periodontal structures.\u003c/p\u003e","manuscriptTitle":"Bio3D graft engineered from human mesenchymal stromal cells/extracellular complexes facilitate periodontal tissue regeneration","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-24 15:29:48","doi":"10.21203/rs.3.rs-7953573/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-04-13T12:35:33+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-30T05:22:52+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"13881425267351757517653317162211883791","date":"2026-03-24T01:37:46+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-01T17:43:07+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"332824448365039959203374838072234399","date":"2026-02-19T20:17:07+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-02-19T17:04:18+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-11-06T23:22:40+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-11-03T16:19:39+00:00","index":"","fulltext":""},{"type":"submitted","content":"npj Regenerative Medicine","date":"2025-10-27T01:06:57+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"npj-regenerative-medicine","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"npjregenmed","sideBox":"Learn more about [npj Regenerative Medicine](http://www.nature.com/npjregenmed/)","snPcode":"41536","submissionUrl":"https://mts-npjregenmed.nature.com/cgi-bin/main.plex","title":"npj Regenerative Medicine","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"NPJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"5e5fe4ec-82dc-4eb1-bfd8-7c4bcb7350fb","owner":[],"postedDate":"February 24th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"in-revision","subjectAreas":[{"id":63223279,"name":"Biological sciences/Biotechnology"},{"id":63223280,"name":"Biological sciences/Cell biology"},{"id":63223281,"name":"Physical sciences/Materials science"},{"id":63223282,"name":"Health sciences/Medical research"},{"id":63223283,"name":"Biological sciences/Stem cells"}],"tags":[],"updatedAt":"2026-04-13T12:41:33+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-24 15:29:48","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7953573","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7953573","identity":"rs-7953573","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}