Calvarial Defect Model Research Articles

Nicotinamide mononucleotide based hyaluronic acid methacryloyl hybrid hydrogel regulating stem cells fate for bone regeneration via SIRT1/RUNX2 signaling

Efficient bone reconstruction, especially of the critical size after bone damage, remains a challenge in the clinic. Bone marrow mesenchymal stem cell (BMSC) osteogenic differentiation is considered as a promising strategy for bone repair. Nicotinamide adenine dinucleotide (NAD+) regulating BMSC fate and cellular function enhance osteogenesis, but is hardly delivered and lack of targeting. Herein, a novel and biocompatible scaffold was fabricated to locally deliver a precursor of NAD+, nicotinamide mononucleotide (NMN) to the bone defect site, and its bone repair capability and healing mechanism were clarified. NMN-based hyaluronic acid methacryloyl hybrid hydrogel scaffold (denoted as NMN/HAMA) was prepared via photopolymerization. In vitro RT-qPCR analysis, western blotting, Elisa and alizarin red S staining assays demonstrated that the NMN/HAMA hybrid hydrogel regulated BMSCs cellular function in favour of osteogenic differentiation and mineralization by upregulating the mRNA and proteins expression of the osteogenic genes type I pro-collagen (Col-1), bone morphogenic protein 4 (BMP4), and runt-related transcription factor 2 (RUNX2) via the SIRT1 pathway. Implantation of such hybrid hydrogels significantly enhanced bone regeneration in rodent critical calvarial defect models. Furthermore, restoration of the bone defect with NMN administration was inhibited in Prx1 Cre+; SIRT1flox/flox mice, confirming that the NMN/HAMA hybrid hydrogel scaffold promoted bone regeneration via the SIRT1-RUNX2 pathway. These results imply that NMN-based scaffold may be a promising and economic strategy for the treatment of bone defects.

Exploring the Osteogenic Potential of Zinc-Doped Magnesium Phosphate Cement (ZMPC): A Novel Material for Orthopedic Bone Defect Repair.

In orthopedics, the repair of bone defects remains challenging. In previous research reports, magnesium phosphate cements (MPCs) were widely used because of their excellent mechanical properties, which have been widely used in the field of orthopedic medicine. We built a new k-struvite (MPC) cement obtained from zinc oxide (ZnO) and assessed its osteogenic properties. Zinc-doped magnesium phosphate cement (ZMPC) is a novel material with good biocompatibility and degradability. This article summarizes the preparation method, physicochemical properties, and biological properties of ZMPC through research on this material. The results show that ZMPC has the same strength and toughness (25.3 ± 1.73 MPa to 20.18 ± 2.11 MPa), that meet the requirements of bone repair. Furthermore, the material can gradually degrade (12.27% ± 1.11% in 28 days) and promote osteogenic differentiation (relative protein expression level increased 2-3 times) of rat bone marrow mesenchymal stem cells (rBMSCs) in vitro. In addition, in vivo confirmation revealed increased bone regeneration in a rat calvarial defect model compared with MPC alone. Therefore, ZMPC has broad application prospects and is expected to be an important repair material in the field of orthopedic medicine.

Osteoimmunomodulatory bioinks for 3D bioprinting achieve complete regeneration of critical-sized bone defects

Regeneration the critical-sized bone defects remains a great challenge to clinical therapy due to the inflammatory microenvironment and lack of stem cells in the region of the bone defects. 3D bioprinted scaffolds based on bioactive ink and loaded active cells can promote the inflammatory microenvironment and cell viability, thereby enhancing bone regeneration. In this study, 10 % Gelatin-methacryloyl (GelMA)/5 %Sr substituted xonotlite (Sr-CSH) nanocomposite hydrogel was developed as a bioink to encapsulate bone marrow mesenchymal stem cells (BMSCs), and then constructed a biomimetic bone tissue by 3D bioprinting. The incorporation of Sr-CSH nanowires enhanced the printing accuracy and mechanical property of GelMA, and enhanced the osteogenic differentiation of BMSCs. In addition, Sr-CSH induced macrophage M2 polarization, which modulated the inflammatory microenvironment and further promoted osteogenic differentiation of BMSCs. In rat critical-sized calvarial defects model, 3D bioprinted scaffolds based on GelMA-Sr-CSH bioinks laden with BMSCs achieve complete bone repair. In summary, this study developed an osteoimmunomodulatory bioink, and 3D bioprinted scaffolds laden with stem cells may be an effective method for achieving complete regeneration of critical-sized bone defects.

Ligand-Selective Targeting of Macrophage Hydrogel Elicits Bone Immune-Stem Cell Endogenous Self-Healing Program to Promote Bone Regeneration.

Targeting macrophages can facilitate the site-specific repair of critical bone defects. Herein, a composite hydrogel, gelatin-Bletilla striata polysaccharide-mesoporous bioactive glass hydrogel (GBMgel), is constructed via the self-assembly of mesoporous bioactive glass on polysaccharide structures, through the Schiff base reaction. GBMgel can efficiently capture macrophages and drive the recruitment of seed stem cells and vascular budding required for regeneration in the early stages of bone injury, and the observed sustained release of inorganic silicon ions further enhances bone matrix deposition, mineralization, and vascular maturation. Moreover, the use of macrophage-depleted rat calvarial defect models further confirms that GBMgel, with ligand-selective macrophage targeting, increases the bone regeneration area and the proportion of mature bone. Mechanistic studies reveal that GBMgel upregulates the TLR4/NF-κB and MAPK macrophage pathways in the early stages and the JAK/STAT3 pathway in the later stages; thus initiating macrophage polarization at different time points. In conclusion, this study is based on the endogenous self-healing properties of bone macrophages, which enhances stem cell homing, and provides a research and theoretical basis upon which bone tissue can be reshaped and regenerated using the body's immune power, providing a new strategy for the treatment of critical bone defects.

Nano-laponite encapsulated coaxial fiber scaffold promotes endochondral osteogenesis.

Osteoinductive supplements without side effects stand out from the growth factors and drugs widely used in bone tissue engineering. Lithium magnesium sodium silicate hydrate (laponite) nanoflake is a promising bioactive component for bone regeneration, attributed to its inherent biosafety and effective osteoinductivity. Up to now, the in vivo osteogenic potential and mechanisms of laponite-encapsulated fibrous membranes remain largely unexplored. This study presents a unique method for homogeneously integrating high concentrations of laponite RDS into a polycaprolactone (PCL) matrix by dispersing laponite RDS sol into the polymer solution. Subsequently, a core-shell fibrous membrane (10RP-PG), embedding laponite-loaded PCL in its core, was crafted using coaxial electrospinning. The PCL core's slow degradation and the shell's gradient degradation enabled the sustained release of bioactive ions (Si and Mg) from laponite. In vivo studies on a critical-sized calvarial bone defect model demonstrated that the 10RP-PG membrane markedly enhanced bone formation and remodeling by accelerating the process of endochondral ossification. Further transcriptome analysis suggested that osteogenesis in the 10RP-PG membrane is driven by Mg and Si from endocytosed laponite, activating pathways related to ossification and endochondral ossification, including Hippo, Wnt and Notch. The fabricated nanocomposite fibrous membranes hold great promise in the fields of critical-sized bone defect repair.

Rapid fabrication of biomimetic PLGA microsphere incorporated with natural porcine dermal aECM for bone regeneration.

Bioactive microspheres coated with acellular extracellular matrix (aECM) have received extensive attention in bone tissue engineering. In this work, biomimetic microspheres with different aECM ratios, uniform size and controllable size were prepared easily by blending natural porcine dermal aECM and poly (lactic-co-glycolic acid) (PLGA) using electrohydrodynamic spraying and solidification actuated by solvent extraction method. In this work, the appropriate polymer concentration and preparation voltage were investigated, and the surface morphology of the microspheres was observed by scanning electron microscope. Sirius red was used to visualize aECM exposure on the surface of the microspheres. The in vitro and in vivo experiments were carried out to evaluate the bioactivity and osteogenic properties of the microspheres. The results showed that the morphology and size of PLGA microspheres had little influence on the aECM blending. In vitro experiments showed that the higher the content of aECM, the better the cell adhesion performance. In vivo, rat calvarial defect models were observed and characterized at 4 and 8 weeks postoperatively, and the values of BV/TV of 50aECM/PLGA were 47.57 ± 1.14% and 72.92 ± 2.19%, respectively. The results showed that the skull healing effect was better in aECM-containing microspheres. In conclusion, aECM/PLGA composite microspheres can increase cell adhesion performance through the addition of aECM. Moreover, in vivo experiments have proved that aECM/PLGA microspheres are beneficial to bone repair, which means the aECM/PLGA microspheres are a promising bone tissue engineering material.

A new hydroxyapatite-alginate-gelatin biocomposite favor bone regeneration in a critical-sized calvarial defect model.

This study aimed to evaluate the osteogenic potential of hydroxyapatite (HA), Alginate (Alg), and Gelatine (Gel) composite in a critical-size defect model in rats. Twenty-four male rats were divided into three groups: a negative control with no treatment (Control group), a positive control treated with deproteinized bovine bone mineral (DBBM group), and the experimental group treated with the new HA-Alg-Gel composite (HA-Alg-Gel group). A critical size defect (8.5mm) was made in the rat's calvaria, and the bone formation was evaluated by in vivo microcomputed tomography analysis (µCT) after 1, 15, 45, and 90 days. After 90 days, the animals were euthanized and histological and histomorphometric analyses were performed. A higher proportion of mineralized tissue/biomaterial was observed in the DBBM group when compared to the HA-Alg-Gel and Control groups in the µCT analysis during all analysis periods. However, no differences were observed in the mineralized tissue/biomaterial proportion observed on day 1 (immediate postoperative) in comparison to later periods of analysis in all groups. In the histomorphometric analysis, the HA-Alg-Gel and Control groups showed higher bone formation than the DBBM group. Moreover, in histological analysis, five samples of the HA-Alg-Gal group exhibited formed bone spicules adjacent to the graft granules against only two of eight samples in the DBBM group. Both graft materials ensured the maintenance of defect bone thickness, while a tissue thickness reduction was observed in the control group. In conclusion, this study demonstrated the osteoconductive potential of HA-Alg-Gel bone graft by supporting new bone formation around its particles.

Exosomes derived from mucoperiosteum Krt14+Ctsk+ cells promote bone regeneration by coupling enhanced osteogenesis and angiogenesis.

Repair of large bone defects is a sophisticated physiological process involving the meticulous orchestration of cell activation, proliferation, and differentiation. Cellular interactions between different cell types are paramount for successful bone regeneration, making it a challenging yet fascinating area of research and clinical practice. With increasing evidence underscoring the essential role of exosomes in facilitating intercellular and cell-microenvironment communication, they have emerged as an encouraging therapeutic strategy to promote bone repair due to their non-immunogenicity, diverse sources, and potent bioactivity. In this study, we characterized a distinctive population of Krt14+Ctsk+ cells from the orbital mucoperiosteum. In vitro experiments confirmed that exosomes from Krt14+Ctsk+ cells dramatically boosted the capacities of human umbilical vein endothelial cells (HUVECs) to proliferate, migrate, and induce angiogenesis. Additionally, the exosomes notably elevated the expression of osteogenic markers, thereby indicating their potential to augment osteogenic capabilities. Furthermore, in vivo experiments utilizing a rat calvarial defect model verified that exosome-loaded sodium alginate (SA) hydrogels accelerated local vascularized bone regeneration within the defective regions. Collectively, these findings suggest that exosomes secreted by Krt14+Ctsk+ cells offer an innovative method to accelerate bone repair via coupling enhanced osteogenesis and angiogenesis, highlighting the therapeutic potential in bone repair.

Comparison of the biocompatibility and osteogenesis potential of whitlockite and an activin A/BMP2 chimera using a rat calvarial defect model: a pilot study.

The aim of this study was to evaluate bone regeneration using whitlockite (WH) nanoparticles, collagenated bovine bone mineral, and an activin A/BMP2 chimera (AB204) on calvarial defects in rats. This study was conducted using 8 rats. Five-millimeter circular defects were formed on each side of the calvaria. The defects were randomly assigned to 3 groups (BO group: BioOss collagen, BO/WH group: BO + WH, and BO/WH/AB204 group: BO + WH + AB204). After healing periods of 2 and 4 weeks, histological and histomorphometric analyses were performed after sacrifice. The BO/WH/AB204 group showed superior bone healing compared to the other 2 groups (BO and BO/WH). In the BO and BO/WH groups, new bone formation was found in the defect margin. However, in the BO/WH/AB204 group, new bone was observed on the upper and lower surfaces of the grafted area. The new bone area of the BO/WH/AB204 group at 4 weeks was significantly higher than that of the same group at 2 weeks. At 4 weeks, the total augmented area and material area in the BO/WH/AB204 group were significantly lower than the corresponding values at 2 weeks. The BO/WH/AB204 group showed superior results of bone regeneration at 2 and 4 weeks compared to the BO and BO/WH groups. AB204 seems to play an important role in bone regeneration.

Guided bone regeneration of calcium phosphate-coated and strontium ranelate-doped titanium mesh in a rat calvarial defect model.

When applied alone, titanium (Ti) mesh may not effectively block the penetration of soft tissues, resulting in insufficient new bone formation. This study aimed to confer bioactivity and improve bone regeneration by doping calcium phosphate (CaP) precipitation and strontium (Sr) ranelate onto a TiO2 nanotube (TNT) layer on the surface of a Ti mesh. The TNT layer was obtained by anodizing on the Ti mesh, and CaP was formed by cyclic pre-calcification. The final specimens were produced by doping with Sr ranelate. The surface properties of the modified Ti mesh were investigated using high-resolution field emission scanning electron microscopy, energy-dispersive X-ray spectroscopy, and X-ray diffraction. To evaluate the effects of surface treatment on cell viability, osteoblasts were cultured for 1-3 days, and their absorbance was subsequently measured. In an in vivo experiment, critical-size defects were created in rat calvaria (Ф=8 mm). After 5 weeks, the rats were sacrificed (n=4 per group) and bone blocks were taken for micro-computed tomography and histological analysis. After immersing the Sr ranelate-doped Ti mesh in simulated body fluid, the protrusions observed in the initial stage of hydroxyapatite were precipitated as a dense structure. On day 3 of osteoblast culture, cell viability was significantly higher on the pre-calcified Sr ranelate-doped Ti mesh surface than on the untreated Ti mesh surface (P<0.05). In the in vivo experiment, a bony bridge formed between the surrounding basal bone and the new bone under the Sr ranelate-doped Ti mesh implanted in a rat calvarial defect, closing the defect. New bone mineral density (0.91±0.003 g/mm3) and bone volume (29.35±2.082 mm3) significantly increased compared to the other groups (P<0.05). Cyclic pre-calcification of a Ti mesh with a uniform TNT layer increased bioactivity, and subsequent doping with Sr ranelate effectively improved bone regeneration in bone defects.

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

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. 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. 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. Within the limitations of this study, the findings indicate that AB204 may enhance osteogenic potential when used in combination with CM for bone regeneration.

Development of a rapid-shaping and user-friendly membrane with long-lasting space maintenance for guided bone regeneration.

The success of guided bone regeneration (GBR) surgery depends largely on the use of GBR membranes to maintain space for bone regeneration and prevent soft tissue ingrowth. However, currently available commercial degradable GBR membranes are often limited by poor space maintenance ability and require additional suture or nail for fixation. To overcome these limitations, we developed a rapid-shaping, adhesive, and user-friendly GBR membrane (PLGA film-PGN) with long-lasting space maintenance by immersing an electrospun poly(lactide-co-glycolic acid) film in a photo-crosslinkable hydrogel composed of polyethylene glycol diacrylate, gelatin methacryloyl, and nanosilicate (PGN). The PGN hydrogel significantly improved the mechanical strength of the PLGA film-PGN and endowed it with plasticity and adhesive properties, making it more maneuverable. The maximum bending force that the PLGA film-PGN could withstand was over 55 times higher than that of the HEAL ALL film (a commonly used commercial GBR membrane). PLGA film-PGN also promoted the proliferation and osteogenic differentiation of rBMSCs. According to a critical-size rat calvarial defect model, PLGA film-PGN maintained the space within the defect area and significantly enhanced bone formation 4 weeks after the surgery. To conclude, the study provided a novel perspective on GBR membrane design and the multifunctional PLGA film-PGN membrane demonstrated great potential for bone defect reconstruction.

Synergistic effect of ROS-generating polydopamine on drug-induced bone tissue regeneration.

A PHD (prolyl hydroxylase) inhibitor, 1,4-dihydrophenonthrolin-4-one-3-carboxylic acid (1,4-DPCA), is a drug that can artificially promote tissue regeneration by enhancing metabolic activity through the upregulation of hypoxia inducible factor 1 subunit alpha (Hif-1α) under normoxic conditions. This study presents a novel design methodology for a drug delivery system to maximize the regenerative effect of 1,4-DPCA. Specifically, by encapsulating 1,4-DPCA in polydopamine (PDA) that generates reactive oxygen species (ROS), the combined effects of Hif-1α upregulation and the induction of cellular antioxidant defense mechanisms by localized ROS can significantly enhance tissue regeneration. The study confirmed that each material (PDA and 1,4-DPCA) triggers a positive synergistic effect on the regenerative mechanisms. As a result, the use of a PDA drug delivery system loaded with 1,4-DPCA showed approximately six times greater bone regeneration compared to the control (no treatment) in a mouse calvarial defect model.

A submicron forest-like silicon surface promotes bone regeneration by regulating macrophage polarization.

Introduction: Silicon is a major trace element in humans and a prospective supporting biomaterial to bone regeneration. Submicron silicon pillars, as a representative surface topography of silicon-based biomaterials, can regulate macrophage and osteoblastic cell responses. However, the design of submicron silicon pillars for promoting bone regeneration still needs to be optimized. In this study, we proposed a submicron forest-like (Fore) silicon surface (Fore) based on photoetching. The smooth (Smo) silicon surface and photoetched regular (Regu) silicon pillar surface were used for comparison in the bone regeneration evaluation. Methods: Surface parameters were investigated using a field emission scanning electron microscope, atomic force microscope, and contact angle instrument. The regulatory effect of macrophage polarization and succedent osteogenesis was studied using Raw264.7, MC3T3-E1, and rBMSCs. Finally, a mouse calvarial defect model was used for evaluating the promoting effect of bone regeneration on the three surfaces. Results: The results showed that the Fore surface can increase the expression of M2-polarized markers (CD163 and CD206) and decrease the expression of inflammatory cytokines, including interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-α). Fore surface can promote the osteogenesis in MC3T3-E1 cells and osteoblastic differentiation of rBMSCs. Furthermore, the volume fraction of new bone and the thickness of trabeculae on the Fore surface were significantly increased, and the expression of RANKL was downregulated. In summary, the upregulation of macrophage M2 polarization on the Fore surface contributed to enhanced osteogenesis in vitro and accelerated bone regeneration in vivo. Discussion: This study strengthens our understanding of the topographic design for developing future silicon-based biomaterials.

Enhanced bone regeneration by osteoinductive and angiogenic zein/whitlockite composite scaffolds loaded with levofloxacin.

Promoting angiogenesis following biomaterial implantation is essential to bone tissue regeneration. Herein, the composite scaffolds composed of zein, whitlockite (WH), and levofloxacin (LEVO) were fabricated to augment bone repair by facilitating osteogenesis and angiogenesis. First, three-dimensional composite scaffolds containing zein and WH were prepared using the salt-leaching method. Then, as a model antibiotic drug, the LEVO was loaded into zein/WH scaffolds. Moreover, the addition of WH enhanced the adhesion, differentiation, and mineralization of osteoblasts. The zein/WH/LEVO composite scaffolds not only had significant osteoinductivity but also showed excellent antibacterial properties. The prepared composite scaffolds were then implanted into a calvarial defect model to evaluate their osteogenic induction effects in vivo. Micro-CT observation and histological analysis indicate that the scaffolds can accelerate bone regeneration with the contribution of endogenous cytokines. Based on amounts of data in vitro and in vivo, the scaffolds present profound effects on improving bone regeneration, especially for the favorable osteogenic, intensive angiogenic, and alleviated inflammation abilities. The results showed that the synthesized scaffolds could be a potential material for bone tissue engineering.

3D printed polylactic acid-based nanocomposite scaffold stuffed with microporous simvastatin-loaded polyelectrolyte for craniofacial reconstruction

Critical sized craniofacial defects are among the most challenging bone defects to repair, due to the anatomical complexity and aesthetic importance. In this study, a polylactic acid/hardystonite-graphene oxide (PLA/HTGO) scaffold was fabricated through 3D printing. In order to upgrade the 3D printed scaffold to a highly porous scaffold, its channels were filled with pectin-quaternized chitosan (Pec-QCs) polyelectrolyte solution containing 0 or 20 mg/mL of simvastatin (Sim) and then freeze-dried. These scaffolds were named FD and FD-Sim, respectively. Also, similar PLA/HTGO scaffolds were prepared and dip coated with Pec-QCs solution containing 0 or 20 mg/mL of Sim and were named DC and DC-Sim, respectively. The formation of macro/microporous structure was confirmed by morphological investigations. The release of Sim from DC-Sim and FD-Sim scaffolds after 28 days was measured as 77.40 ± 5.25 and 86.02 ± 3.63 %, respectively. Cytocompatibility assessments showed that MG-63 cells had the highest proliferation, attachment and spread on the Sim containing scaffolds, especially FD-Sim. In vivo studies on a rat calvarial defect model revealed that an almost complete recovery occurred in the group treated with FD-Sim scaffold after 8 weeks and the defect was filled with newly formed bone. The results of this study acknowledge that the FD-Sim scaffold can be a perfect candidate for calvarial defect repair.

Bone Regeneration Potential of Periodontal Ligament Stem Cells in Combination with Cold Atmospheric Plasma-Pretreated Beta-Tricalcium Phosphate: An In Vivo Assessment

In regenerative bone tissue medicine, combining artificial bone substitutes with progenitor cells is a prospective approach. Surface modification via cold atmospheric plasma (CAP) enhances biomaterial–cell interactions, which are crucial for successful bone regeneration. Using a rabbit calvarial critical-size defect model, we assessed the use of CAP-pretreated beta-tricalcium phosphate (β-TCP), alone or with periodontal ligament stem cells (PDLSCs), for bone regeneration. Histological and histomorphometric analyses at two and four weeks revealed significantly improved bone regeneration and reduced inflammation in the CAP-treated β-TCP with PDLSCs compared to β-TCP alone. Immunohistochemical analysis also showed an increase in the bone healing markers, including bone morphogenic proteins 2 and 4, runt-related transcription factor 2, collagen-1, and osteonectin, after two and four weeks in the CAP-treated β-TCP implants with PDLSC. This in vivo study demonstrates for the first time the superior bone regenerative capacity of CAP-pretreated β-TCP seeded with PDLSCs, highlighting the therapeutic potential of this combined approach in osteoregeneration.

Enhanced bone regeneration via endochondral ossification using Exendin-4-modified mesenchymal stem cells

Nonunions and delayed unions pose significant challenges in orthopedic treatment, with current therapies often proving inadequate. Bone tissue engineering (BTE), particularly through endochondral ossification (ECO), emerges as a promising strategy for addressing critical bone defects. This study introduces mesenchymal stem cells overexpressing Exendin-4 (MSC-E4), designed to modulate bone remodeling via their autocrine and paracrine functions. We established a type I collagen (Col-I) sponge-based in vitro model that effectively recapitulates the ECO pathway. MSC-E4 demonstrated superior chondrogenic and hypertrophic differentiation and enhanced the ECO cell fate in single-cell sequencing analysis. Furthermore, MSC-E4 encapsulated in microscaffold, effectively facilitated bone regeneration in a rat calvarial defect model, underscoring its potential as a therapeutic agent for bone regeneration. Our findings advocate for MSC-E4 within a BTE framework as a novel and potent approach for treating significant bone defects, leveraging the intrinsic ECO process.

Targeting class A GPCRs for hard tissue regeneration

G protein-coupled receptors (GPCRs) play important roles in various pathogeneses and physiological regulations. Owing to their functional diversity, GPCRs are considered one of the primary pharmaceutical targets. However, drugs targeting GPCRs have not been developed yet to regenerate hard tissues such as teeth and bones. Mesenchymal stromal cells (MSCs) have high proliferation and multi-lineage differentiation potential, which are essential for hard tissue regeneration. Here, we present a strategy for targeting class A GPCRs for hard tissue regeneration by promoting the differentiation of endogenous MSCs into osteogenic and odontogenic progenitor cells. Through in vitro screening targeted at class A GPCRs, we identified six target receptors (LPAR1, F2R, F2RL1, F2RL2, S1PR1, and ADORA2A) and candidate drugs with potent biomineralization effects. Through a combination of profiling whole transcriptome and accessible chromatin regions, we identified that p53 acts as a key transcriptional activator of genes that modulate the biomineralization process. Moreover, the therapeutic potential of class A GPCR-targeting drugs was demonstrated in tooth pulpotomy and calvarial defect models. The selected drugs revealed potent regenerative effects in both tooth and bone defects, represented by newly formed highly mineralized regions. Consequently, this study provides translational evidence for a new regenerative strategy for damaged hard tissue.

Multimodality imaging reveals angiogenic evolution in vivo during calvarial bone defect healing.

The healing of calvarial bone defects is a pressing clinical problem that involves the dynamic interplay between angiogenesis and osteogenesis within the osteogenic niche. Although structural and functional vascular remodeling (i.e., angiogenic evolution) in the osteogenic niche is a crucial modulator of oxygenation, inflammatory and bone precursor cells, most clinical and pre-clinical investigations have been limited to characterizing structural changes in the vasculature and bone. Therefore, we developed a new multimodality imaging approach that for the first time enabled the longitudinal (i.e., over four weeks) and dynamic characterization of multiple in vivo functional parameters in the remodeled vasculature and its effects on de novo osteogenesis, in a preclinical calvarial defect model. We employed multi-wavelength intrinsic optical signal (IOS) imaging to assess microvascular remodeling, intravascular oxygenation (SO2), and osteogenesis; laser speckle contrast (LSC) imaging to assess concomitant changes in blood flow and vascular maturity; and micro-computed tomography (μCT) to validate volumetric changes in calvarial bone. We found that angiogenic evolution was tightly coupled with calvarial bone regeneration and corresponded to distinct phases of bone healing, such as injury, hematoma formation, revascularization, and remodeling. The first three phases occurred during the initial two weeks of bone healing and were characterized by significant in vivo changes in vascular morphology, blood flow, oxygenation, and maturity. Overall, angiogenic evolution preceded osteogenesis, which only plateaued toward the end of bone healing (i.e., four weeks). Collectively, these data indicate the crucial role of angiogenic evolution in osteogenesis. We believe that such multimodality imaging approaches have the potential to inform the design of more efficacious tissue-engineering calvarial defect treatments.