Fundamentals and Translational Applications of Stem Cells and Biomaterials in Dental, Oral and Craniofacial Regenerative Medicine.

Regenerative dental medicine continuously expands to improve treatments for prevalent clinical problems in dental and oral medicine. Stem cell based translational opportunities include regenerative therapies for tooth restoration, root canal therapy, and inflammatory processes (e.g., periodontitis). The potential of regenerative approaches relies on the biological properties of dental stem cells. These and other multipotent somatic mesenchymal stem cell (MSC) types can in principle be applied as either autologous or allogeneic sources in dental procedures. Dental stem cells have distinct developmental origins and biological markers that determine their translational utility. Dental regenerative medicine is supported by mechanistic knowledge of the molecular pathways that regulate dental stem cell growth and differentiation. Cell fate determination and lineage progression of dental stem cells is regulated by multiple cell signaling pathways (e.g., WNTs, BMPs) and epigenetic mechanisms, including DNA modifications, histone modifications, and non-coding RNAs (e.g., miRNAs and lncRNAs). This review also considers a broad range of novel approaches in which stem cells are applied in combination with biopolymers, ceramics, and composite materials, as well as small molecules (agonistic or anti-agonistic ligands) and natural compounds. Materials that mimic the microenvironment of the stem cell niche are also presented. Promising concepts in bone and dental tissue engineering continue to drive innovation in dental and non-dental restorative procedures.

Evaluation of alveolar ridge preservation in sockets with buccal dehiscence defects using two types of xenogeneic biomaterials: An in vivo experimental study.

OBJECTIVES: Alveolar ridge preservation (ARP) has been extensively investigated in various preclinical and clinical studies, yielding favorable results. We aim to evaluate the effects of ARP using collagenated bovine bone mineral (CBBM) alone or particulated bovine bone mineral with a non-cross-linked collagen membrane (PBBM/NCLM) in tooth extraction sockets with buccal dehiscence in an experimental dog model. MATERIALS AND METHODS: The mesial roots of three mandibular premolars (P2, P3, and P4) were extracted from six mongrel dogs 4 weeks after inducing dehiscence defects. ARP was randomly performed using two different protocols: 1) CBBM alone and 2) PBBM/NCLM. Three-dimensional (3D) volumetric, micro-computed tomography, and histological analyses were employed to determine changes over a span of 20 weeks. RESULTS: In 3D volumetric and radiographic analyses, CBBM alone demonstrated similar effectiveness to PBBM/NCLM in ARP (p > .05). However, in the PBBM/NCLM group (3.05 ± 0.60 mm), the horizontal ridge width was well maintained 3 mm below the alveolar crest compared with the CBBM group (2.11 ± 1.01 mm, p = .002). CONCLUSION: Although the radiographic changes in the quality and quantity of bone were not significant between the two groups, the use of PBBM/NCLM resulted in greater horizontal dimensions and more favorable maintenance of the ridge profile.

ROS-induced PADI2 downregulation accelerates cellular senescence via the stimulation of SASP production and NFκB activation.

Cellular senescence is closely related to tissue aging including bone. Bone homeostasis is maintained by the tight balance between bone-forming osteoblasts and bone-resorbing osteoclasts, but it undergoes deregulation with age, causing age-associated osteoporosis, a main cause of which is osteoblast dysfunction. Oxidative stress caused by the accumulation of reactive oxygen species (ROS) in bone tissues with aging can accelerate osteoblast senescence and dysfunction. However, the regulatory mechanism that controls the ROS-induced senescence of osteoblasts is poorly understood. Here, we identified Peptidyl arginine deiminase 2 (PADI2), a post-translational modifying enzyme, as a regulator of ROS-accelerated senescence of osteoblasts via RNA-sequencing and further functional validations. PADI2 downregulation by treatment with H2O2 or its siRNA promoted cellular senescence and suppressed osteoblast differentiation. CCL2, 5, and 7 known as the elements of the senescence-associated secretory phenotype (SASP) which is a secretome including proinflammatory cytokines and chemokines emitted by senescent cells and a representative feature of senescence, were upregulated by H2O2 treatment or Padi2 knockdown. Furthermore, blocking these SASP factors with neutralizing antibodies or siRNAs alleviated the senescence and dysfunction of osteoblasts induced by H2O2 treatment or Padi2 knockdown. The elevated production of these SASP factors was mediated by the activation of NFκB signaling pathway. The inhibition of NFκB using the pharmacological inhibitor or siRNA effectively relieved H2O2 treatment- or Padi2 knockdown-induced senescence and osteoblast dysfunction. Together, our study for the first time uncover the role of PADI2 in ROS-accelerated cellular senescence of osteoblasts and provide new mechanistic and therapeutic insights into excessive ROS-promoted cellular senescence and aging-related bone diseases.

Foci-Xpress: Automated and Fast Nuclear Foci Counting Tool.

In the nucleus, distinct, discrete spots or regions called "foci" have been identified, each harboring a specific molecular function. Accurate and efficient quantification of these foci is essential for understanding cellular dynamics and signaling pathways. In this study, we present an innovative automated image analysis method designed to precisely quantify subcellular foci within the cell nucleus. Manual foci counting methods can be tedious and time-consuming. To address these challenges, we developed an open-source software that automatically counts the number of foci from the indicated image files. We compared the foci counting efficiency, velocity, accuracy, and convenience of Foci-Xpress with those of other conventional methods in foci-induced models. We can adjust the brightness of foci to establish a threshold. The Foci-Xpress method was significantly faster than other conventional methods. Its accuracy was similar to that of conventional methods. The most significant strength of Foci-Xpress is automation, which eliminates the need for analyzing equipment while counting. This enhanced throughput facilitates comprehensive statistical analyses and supports robust conclusions from experiments. Furthermore, automation completely rules out biases caused by researchers, such as manual errors or daily variations. Thus, Foci-Xpress is a convincing, convenient, and easily accessible focus-counting tool for cell biologists.

Surface Topography of Titanium Affects Their Osteogenic Potential through DNA Methylation.

It is widely accepted that sandblasted/large-grit/acid-etched (SLA) surfaces of titanium (Ti) have a higher osteogenic potential than machined ones. However, most studies focused on differential gene expression without elucidating the underlying mechanism for this difference. The aim of this study was to evaluate how the surface roughness of dental Ti implants affects their osteogenic potential. Mouse preosteoblast MC3T3-E1 cells were seeded on machined and SLA Ti discs. The cellular activities of the discs were analyzed using confocal laser scanning microscopy, proliferation assays, and real-time polymerase chain reaction (PCR). DNA methylation was evaluated using a methylation-specific PCR. The cell morphology was slightly different between the two types of surfaces. While cellular proliferation was slightly greater on the machined surfaces, the osteogenic response of the SLA surfaces was superior, and they showed increased alkaline phosphatase (Alp) activity and higher bone marker gene expression levels (Type I collagen, Alp, and osteocalcin). The degree of DNA methylation on the Alp gene was lower on the SLA surfaces than on the machined surfaces. DNA methyltransferase inhibitor stimulated the Alp gene expression on the machined surfaces, similar to the SLA surfaces. The superior osteogenic potential of the SLA surfaces can be attributed to a different epigenetic landscape, specifically, the DNA methylation of Alp genes. This finding offers novel insights into epigenetics to supplement genetics and raises the possibility of using epidrugs as potential therapeutic targets to enhance osteogenesis on implant surfaces.

A randomized controlled clinical trial evaluating efficacy and adverse events of different types of recombinant human bone morphogenetic protein-2 delivery systems for alveolar ridge preservation.

OBJECTIVES: This 12-week clinical trial evaluated efficacy and adverse events for two recombinant human bone morphogenetic protein-2 (rhBMP-2) delivery systems in alveolar ridge preservation. MATERIALS AND METHODS: Sixty-four patients had a single tooth that required replacement with an implant, surrounded by > 50% alveolar bone height. Two cohorts (n = 32 patients each) were randomized to receive a rhBMP-2-soaked absorbable collagen sponge (test group), or ß-tricalcium phosphate and hydroxyapatite particles (control group) immersed in rhBMP-2, at the implant site. Bone height and width changes at 25%, 50%, and 75% of extraction socket level (ESL) were compared. Adverse events were assessed in the same period. In addition to the randomized controlled clinical trial, histological analysis of 21 patients (test group [n = 12], control group [n = 9]) was conducted, 4 months after alveolar ridge preservation. A non-inferiority test was used to analyze changes in alveolar bone height between groups (p = 0.05). A Wilcoxon rank-sum test was used to analyze changes in alveolar bone width and histomorphometric results between groups (p = 0.05). RESULTS: All patients showed good healing without severe adverse events. The lower limit of the one-sided 97.5% confidence interval in the difference between the two groups was 0.0033 (non-inferiority margin: -0.185); thus, the test group showed non-inferiority to the control group. Wilcoxon rank-sum test analysis did not show statistically significant differences between groups with regard to changes in alveolar bone width and histomorphometric analysis. CONCLUSIONS: The delivery systems showed similar efficacy for alveolar ridge preservation without severe adverse events.

Pin1-mediated Modification Prolongs the Nuclear Retention of ß-Catenin in Wnt3a-induced Osteoblast Differentiation.

The canonical Wnt signaling pathway, in which ß-catenin nuclear localization is a crucial step, plays an important role in osteoblast differentiation. Pin1, a prolyl isomerase, is also known as a key enzyme in osteogenesis. However, the role of Pin1 in canonical Wnt signal-induced osteoblast differentiation is poorly understood. We found that Pin1 deficiency caused osteopenia and reduction of ß-catenin in bone lining cells. Similarly, Pin1 knockdown or treatment with Pin1 inhibitors strongly decreased the nuclear ß-catenin level, TOP flash activity, and expression of bone marker genes induced by canonical Wnt activation and vice versa in Pin1 overexpression. Pin1 interacts directly with and isomerizes ß-catenin in the nucleus. The isomerized ß-catenin could not bind to nuclear adenomatous polyposis coli, which drives ß-catenin out of the nucleus for proteasomal degradation, which consequently increases the retention of ß-catenin in the nucleus and might explain the decrease of ß-catenin ubiquitination. These results indicate that Pin1 could be a critical target to modulate ß-catenin-mediated osteogenesis.

Direct Delivery of Recombinant Pin1 Protein Rescued Osteoblast Differentiation of Pin1-Deficient Cells.

Pin1 is a peptidyl prolyl cis-trans isomerase that specifically binds to the phosphoserine-proline or phosphothreonine-proline motifs of several proteins. We reported that Pin1 plays a critical role in the fate determination of Smad1/5, Runx2, and ß-catenin that are indispensable nuclear proteins for osteoblast differentiation. Though several chemical inhibitors has been discovered for Pin1, no activator has been reported as of yet. In this study, we directly introduced recombinant Pin1 protein successfully into the cytoplasm via fibroin nanoparticle encapsulated in cationic lipid. This nanoparticle-lipid complex delivered its cargo with a high efficiency and a low cytotoxicity. Direct delivery of Pin1 leads to increased Runx2 and Smad signaling and resulted in recovery of the osteogenic marker genes expression and the deposition of mineral in Pin1-deficient cells. These result indicated that a direct Pin1 protein delivery method could be a potential therapeutics for the osteopenic diseases. J. Cell. Physiol. 232: 2798-2805, 2017. © 2016 Wiley Periodicals, Inc.

Epigenetic Priming Confers Direct Cell Trans-Differentiation From Adipocyte to Osteoblast in a Transgene-Free State.

The bone marrow of healthy individuals is primarily composed of osteoblasts and hematopoietic cells, while that of osteoporosis patients has a larger portion of adipocytes. There is evidence that the epigenetic landscape can strongly influence cell differentiation. We have shown that it is possible to direct the trans-differentiation of adipocytes to osteoblasts by modifying the epigenetic landscape with a DNA methyltransferase inhibitor (DNMTi), 5'-aza-dC, followed by Wnt3a treatment to signal osteogenesis. Treating 3T3-L1 adipocytes with 5'-aza-dC induced demethylation in the hypermethylated CpG regions of bone marker genes; subsequent Wnt3a treatment drove the cells to osteogenic differentiation. When old mice with predominantly adipose marrow were treated with both 5'-aza-dC and Wnt3a, decreased fatty tissue and increased bone volume were observed. Together, our results indicate that epigenetic modification permits direct programming of adipocytes into osteoblasts in a mouse model of osteoporosis, suggesting that this approach could be useful in bone tissue-engineering applications.

Epigenetic modifications and canonical wingless/int-1 class (WNT) signaling enable trans-differentiation of nonosteogenic cells into osteoblasts.

Mesenchymal cells alter and retain their phenotype during skeletal development through activation or suppression of signaling pathways. For example, we have shown that Wnt3a only stimulates osteoblast differentiation in cells with intrinsic osteogenic potential (e.g. MC3T3-E1 pre-osteoblasts) and not in fat cell precursors or fibroblasts (3T3-L1 pre-adipocytes or NIH3T3 fibroblasts, respectively). Wnt3a promotes osteogenesis in part by stimulating autocrine production of the osteoinductive ligand Bmp2. Here, we show that the promoter regions of the genes for Bmp2 and the osteoblast marker Alp are epigenetically locked to prevent their expression in nonosteogenic cells. Both genes have conserved CpG islands that exhibit increased CpG methylation, as well as decreased acetylation and increased methylation of histone H3 lysine 9 (H3-K9) specifically in nonosteogenic cells. Treatment of pre-adipocytes or fibroblasts with the CpG-demethylating agent 5'-aza-2'-deoxycytidine or the histone deacetylase inhibitor trichostatin-A renders Bmp2 and Alp responsive to Wnt3a. Hence, drug-induced epigenetic activation of Bmp2 gene expression contributes to Wnt3a-mediated direct trans-differentiation of pre-adipocytes or fibroblasts into osteoblasts. We propose that direct conversion of nonosteogenic cells into osteoblastic cell types without inducing pluripotency may improve prospects for novel epigenetic therapies to treat skeletal afflictions.

Prolyl isomerase Pin1-mediated conformational change and subnuclear focal accumulation of Runx2 are crucial for fibroblast growth factor 2 (FGF2)-induced osteoblast differentiation.

Fibroblast growth factor 2 (FGF2) signaling plays a pivotal role in bone growth/differentiation through the activation of osteogenic master transcription factor Runx2, which is mediated by the ERK/MAPK-dependent phosphorylation and the p300-dependent acetylation of Runx2. In this study, we found that Pin1-dependent isomerization of Runx2 is the critical step for FGF2-induced Runx2 transactivation function. We identified four serine or threonine residues in the C-terminal domain of Runx2 that are responsible for Pin1 binding and structural modification. Confocal imaging studies indicated that FGF2 treatment strongly stimulated the focal accumulation of Pin1 in the subnuclear area, which recruited Runx2. In addition, active forms of RNA polymerase-II also colocalized in the same subnuclear compartment. Dipentamethylene thiuram monosulfide, a Pin1 inhibitor, strongly attenuated their focal accumulation as well as Runx2 transactivation activity. The Pin1-mediated structural modification of Runx2 is an indispensable step connecting phosphorylation and acetylation and, consequently, transcriptional activation of Runx2 by FGF signaling. Thus, the modulation of Pin1 activity may be a target for the regulation of bone formation.

Pin1 plays a critical role as a molecular switch in canonical BMP signaling.

Pin1 is a peptidyl prolyl cis-trans isomerase that specifically binds to the phosphoserine-proline or phosphothreonine-proline motifs of numerous proteins. Previously, we reported that Pin1 deficiency resulted in defects in osteoblast differentiation during early bone development. In this study, we found that adult Pin1-deficient mice developed osteoporotic phenotypes compared to age-matched controls. Since BMP2 stored in the bone matrix plays a critical role in adult bone maintenance, we suspected that BMP R-Smads (Smad1 and Smad5) could be critical targets for Pin1 action. Pin1 specifically binds to the phosphorylated linker region of Smad1, which leads to structural modification and stabilization of the Smad1 protein. In this process, Pin1-mediated conformational modification of Smad1 directly suppresses the Smurf1 interaction with Smad1, thereby promoting sustained activation of the Smad1 molecule. Our data demonstrate that post-phosphorylational prolyl isomerization of Smad1 is a converging signal to stabilize the Smad1 molecule against the ubiquitination process mediated by Smurf1. Therefore, Pin1 is a critical molecular switch in the determination of Smad1 fate, opposing the death signal transmitted to the Smad1 linker region by phosphorylation cascades after its nuclear localization and transcriptional activation. Thus, Pin1 could be developed as a major therapeutic target in many skeletal diseases.

Comparison of the osteogenic potential of titanium- and modified zirconia-based bioceramics.

Zirconia is now favored over titanium for use in dental implant materials because of its superior aesthetic qualities. However, zirconia is susceptible to degradation at lower temperatures. In order to address this issue, we have developed modified zirconia implants that contain tantalum oxide or niobium oxide. Cells attached as efficiently to the zirconia implants as to titanium-based materials, irrespective of surface roughness. Cell proliferation on the polished surface was higher than that on the rough surfaces, but the converse was true for the osteogenic response. Cells on yttrium (Y)/tantalum (Ta)- and yttrium (Y)/niobium (Nb)-stabilized tetragonal zirconia polycrystals (TZP) discs ((Y, Ta)-TZP and (Y, Nb)-TZP, respectively) had a similar proliferative potential as those grown on anodized titanium. The osteogenic potential of MC3T3-E1 pre-osteoblast cells on (Y, Ta)-TZP and (Y, Nb)-TZP was similar to that of cells grown on rough-surface titanium. These data demonstrate that improved zirconia implants, which are resistant to temperature-induced degradation, retain the desirable clinical properties of structural stability and support of an osteogenic response.

Clinical evaluation of 3.0-mm narrow-diameter implants: a retrospective study with up to 5 years of observation.

PURPOSE: This study aimed to evaluate the clinical outcomes of a single type of narrow-diameter implant (NDI) by investigating its survival rate and peri-implant marginal bone loss (MBL). In addition, variables possibly related to implant survival and MBL were investigated to identify potential risk factors. METHODS: The study was conducted as a retrospective study involving 49 patients who had received 3.0-mm diameter TSIII implants (Osstem Implant Co.) at Seoul National University Dental Hospital. In total, 64 implants were included, and dental records and radiographic data were collected from 2017 to 2022. Kaplan-Meier survival curves and a Cox proportional hazard model were used to estimate the implant survival rate and to investigate the effects of age, sex, jaw, implant location, implant length, the stage of surgery, guided bone regeneration, type of implant placement, and the surgeon's proficiency (resident or professor) on implant survival. The MBL of the NDIs was measured, and the factors influencing MBL were evaluated. RESULTS: The mean observation period was 30.5 months (interquartile range, 26.75-45 months), and 6 out of 64 implants failed. The survival rate of the NDIs was 90.6%, and the multivariate Cox regression analysis showed that age was associated with implant failure (hazard ratio, 1.17; 95% confidence interval, 1.04-1.31, P=0.01). The mean MBL was 0.44±0.75 mm, and no factors showed statistically significant associations with greater MBL. CONCLUSIONS: NDIs can be considered a primary alternative when standard-diameter implants are unsuitable. However, further studies are required to confirm their long-term stability.

The activin/BMP-2 chimera AB204 promotes periodontal tissue regeneration in a buccal dehiscence model: a pilot study.

PURPOSE: A combination of activin and bone morphogenetic protein-2 (BMP-2), termed AB204, has been shown to improve osteogenic potential with fewer side effects than BMP-2 alone. This study was performed to evaluate the effect of AB204 on periodontal tissue regeneration in a dog buccal dehiscence model. METHODS: Buccal dehiscence defects were created on the maxillary premolars (P1, P2, and P3) of 6 mongrel dogs. After 5 weeks, the dogs were randomly assigned to 1 of 3 groups: the control, collagen matrix (CM), and CM/AB204 groups. Grafting procedures were then performed. The dogs were sacrificed 8 weeks after the grafting procedure, and volumetric and histological analyses were conducted. RESULTS: The thickness of the buccal gingiva in the CM/AB204 group was greater than those in the other groups at 2 weeks (P<0.05). The ridge width in the AB204/CM group exceeded the width in the other groups at 4 and 8 weeks; however, the difference was not statistically significant. Histological analysis revealed that the CM/AB204 group demonstrated the formation of new bone surrounded by newly formed periodontal ligament and cementum (P=0.035). CONCLUSIONS: The combined application of CM and AB204 shows promise in facilitating the regeneration of periodontal attachment, including the formation of new bone, cementum, and periodontal ligament.

Bone regeneration using activin A/BMP2 chimera (AB204) with collagen membrane in rats with calvarial defects.

PURPOSE: Collagen has long been recognized as an excellent carrier for growth factors, and membrane-type collagen has been widely applied in dentistry for guided bone regeneration. This study was conducted to examine the effects of an activin A/BMP2 chimera (AB204) combined with a collagen membrane (CM) on bone repair in a rat calvarial defect model. METHODS: A unilateral calvarial defect measuring 5.0 mm was surgically created in 32 Sprague-Dawley rats. The rats were then randomly assigned to 1 of 4 groups, each consisting of 8 animals: control (untreated), CM (treated with a CM only), CM/bone morphogenetic protein 2 (BMP2) (treated with a CM and 1.0 µg of BMP2), and CM/AB204 (treated with a CM and 1.0 µg of AB204). Bone regeneration was evaluated using micro-computed tomography (CT) and histological analysis at 2 and 4 weeks following surgery. RESULTS: Micro-CT analysis revealed that bone formation in the CM/BMP2 and CM/AB204 groups was superior to that observed in the control and CM groups at both 2 and 4 weeks postoperatively. BMP2 induced greater bone regeneration than AB204 at 2 weeks; however, AB204 resulted in a greater bone volume at 4 weeks, achieving the highest values recorded. No significant differences were found between the CM/BMP2 and CM/AB204 groups at either time point (P>0.05). On histological examination, new bone formation was evident in both CM/BMP2 and CM/AB204 groups. CONCLUSIONS: Within the limitations of this study, the findings indicate that AB204 may enhance osteogenic potential when used in combination with CM for bone regeneration.

Effectiveness of a collagen matrix seal and xenograft in alveolar ridge preservation: an experimental study in dogs.

Majority of previous studies on alveolar ridge preservation (ARP) used collagen membranes as barrier membranes, and further evidence for ARP in dehiscent extraction sockets with a deproteinized bovine bone mineral (DBBM) and matrix is needed. The aim of this study is to assess the impact of non-cross linked collagen membranes (membrane) and crosslinked collagen matrices (matrix) on ARP using DBBM in extraction sockets with buccal dehiscence. In six mongrel dogs, the mesial roots of three mandibular premolars (P2, P3, and P4) were extracted 1 month after dehiscence defect induction. Two experimental groups were randomly assigned: (1) DBBM with a membrane (DBBM/membrane group) and (2) DBBM with a matrix (DBBM/matrix group). Three-dimensional (3D) volumetric, microcomputed tomography (µCT), and histologic analyses were performed to assess the ridge preservation. Both groups were effective to maintain the ridge width (p > 0.05), and the DBBM/matrix group showed more favorable soft tissue regeneration and bone quality in the histological analysis (p = 0.05). Based on these results, DBBM/matrix could be better choice for ARP in cases of buccal dehiscence defects.

The cumulative survival rate of dental implants with micro-threads: a long-term retrospective study.

PURPOSE: This study aimed to evaluate the long-term cumulative survival rate (CSR) of dental implants with micro-threads in the neck over a 10-year follow-up period and to examine the factors influencing the survival rate of dental implants. METHODS: This retrospective study was based on radiographic and dental records. In total, 151 patients received 490 Oneplant® dental implants with an implant neck micro-thread design during 2006-2010 in the Department of Periodontology of Seoul National University Dental Hospital. Implant survival was evaluated using Kaplan-Meier analysis. Cox proportional hazard regression analysis was used to identify the factors influencing implant failure. RESULTS: Ten out of 490 implants (2.04%) failed due to fixture fracture. The CSR of the implants was 97.9%, and no significant difference was observed in the CSR between external- and internal-implant types (98.2% and 97.6%, respectively, P=0.670). In Cox regression analysis, 2-stage surgery significantly increased the risk of implant failure (hazard ratio: 4.769, P=0.039). There were no significant differences in influencing factors, including sex, age, implant diameter, length, fixture type, location, surgical procedure, bone grafting, and restoration type. CONCLUSIONS: Within the limitations of this retrospective study, the micro-thread design of the implant neck was found to be favorable for implant survival, with stable clinical outcomes.

Bone-targeted lipoplex-loaded three-dimensional bioprinting bilayer scaffold enhanced bone regeneration.

Clinical bone-morphogenetic protein 2 (BMP2) treatment for bone regeneration, often resulting in complications like soft tissue inflammation and ectopic ossification due to high dosages and non-specific delivery systems, necessitates research into improved biomaterials for better BMP2 stability and retention. To tackle this challenge, we introduced a groundbreaking bone-targeted, lipoplex-loaded, three-dimensional bioprinted bilayer scaffold, termed the polycaprolactone-bioink-nanoparticle (PBN) scaffold, aimed at boosting bone regeneration. We encapsulated BMP2 within the fibroin nanoparticle based lipoplex (Fibroplex) and functionalized it with DSS6 for bone tissue-specific targeting. 3D printing technology enables customized, porous PCL scaffolds for bone healing and soft tissue growth, with a two-step bioprinting process creating a cellular lattice structure and a bioink grid using gelatin-alginate hydrogel and DSS6-Fibroplex, shown to support effective nutrient exchange and cell growth at specific pore sizes. The PBN scaffold is predicted through in silico analysis to exhibit biased BMP2 release between bone and soft tissue, a finding validated by in vitro osteogenic differentiation assays. The PBN scaffold was evaluated for critical calvarial defects, focusing on sustained BMP2 delivery, prevention of soft tissue cell infiltration and controlled fiber membrane pore size in vivo. The PBN scaffold demonstrated a more than eight times longer BMP2 release time than that of the collagen sponge, promoting osteogenic differentiation and bone regeneration in a calvarial defect animal. Our findings suggest that the PBN scaffold enhanced the local concentration of BMP2 in bone defects through sustained release and improved the spatial arrangement of bone formation, thereby reducing the risk of heterotopic ossification.

Pin1-mediated Runx2 modification is critical for skeletal development.

Runx2 is the master transcription factor for bone formation. Haploinsufficiency of RUNX2 is the genetic cause of cleidocranial dysplasia (CCD) that is characterized by hypoplastic clavicles and open fontanels. In this study, we found that Pin1, peptidyl prolyl cis-trans isomerase, is a critical regulator of Runx2 in vivo and in vitro. Pin1 mutant mice developed CCD-like phenotypes with hypoplastic clavicles and open fontanels as found in the Runx2+/- mice. In addition Runx2 protein level was significantly reduced in Pin1 mutant mice. Moreover Pin1 directly interacts with the Runx2 protein in a phosphorylation-dependent manner and subsequently stabilizes Runx2 protein. In the absence of Pin1, Runx2 is rapidly degraded by the ubiquitin-dependent protein degradation pathway. However, Pin1 overexpression strongly attenuated uniquitin-dependent Runx2 degradation. Collectively conformational change of Runx2 by Pin1 is essential for its protein stability and possibly enhances the level of active Runx2 in vivo.