Calvarial Bone Repair Research Articles

Requirement of Pdgfrα+ cells for calvarial bone repair.

Platelet-derived growth factor receptor α (PDGFRα) is often considered as a general marker of mesenchymal cells and fibroblasts, but also shows expression in a portion of osteoprogenitor cells. Within the skeleton, Pdgfrα+ mesenchymal cells have been identified in bone marrow and periosteum of long bones, where they play a crucial role in participating in fracture repair. A similar examination of Pdgfrα+ cells in calvarial bone healing has not been examined. Here, we utilize Pdgfrα-CreERTM;mT/mG reporter animals to examine the contribution of Pdgfrα+ mesenchymal cells to calvarial bone repair through histology and single-cell RNA sequencing (scRNA-Seq). Results showed that Pdgfrα+ mesenchymal cells are present in several cell clusters by scRNA-Seq, and by histology a dramatic increase in Pdgfrα+ cells populated the defect site at early timepoints to give rise to healed bone tissue overtime. Notably, diphtheria toxin-mediated ablation of Pdgfrα reporter+ cells resulted in significantly impaired calvarial bone healing. Our findings suggest that Pdgfrα-expressing cells within the calvarial niche play a critical role in the process of calvarial bone repair.

Angiogenesis is uncoupled from osteogenesis during calvarial bone regeneration

Bone regeneration requires a well-orchestrated cellular and molecular response including robust vascularization and recruitment of mesenchymal and osteogenic cells. In femoral fractures, angiogenesis and osteogenesis are closely coupled during the complex healing process. Here, we show with advanced longitudinal intravital multiphoton microscopy that early vascular sprouting is not directly coupled to osteoprogenitor invasion during calvarial bone regeneration. Early osteoprogenitors emerging from the periosteum give rise to bone-forming osteoblasts at the injured calvarial bone edge. Microvessels growing inside the lesions are not associated with osteoprogenitors. Subsequently, osteogenic cells collectively invade the vascularized and perfused lesion as a multicellular layer, thereby advancing regenerative ossification. Vascular sprouting and remodeling result in dynamic blood flow alterations to accommodate the growing bone. Single cell profiling of injured calvarial bones demonstrates mesenchymal stromal cell heterogeneity comparable to femoral fractures with increase in cell types promoting bone regeneration. Expression of angiogenesis and hypoxia-related genes are slightly elevated reflecting ossification of a vascularized lesion site. Endothelial Notch and VEGF signaling alter vascular growth in calvarial bone repair without affecting the ossification progress. Our findings may have clinical implications for bone regeneration and bioengineering approaches.

Enhanced Calvarial Bone Repair Using ASCs Engineered with RNA-Guided Split dCas12a System that Co-Activates Sox 5, Sox6, and Long Non-Coding RNA H19.

Healing of large calvarial bone defects remains challenging. An RNA-guided Split dCas12a system is previously harnessed to activate long non-coding RNA H19 (lncRNA H19, referred to as H19 thereafter) in bone marrow-derived mesenchymal stemcells (BMSCs). H19 activation in BMSCs induces chondrogenic differentiation, switches bone healing pathways, and improves calvarial bone repair. Since adipose-derived stem cells (ASCs) can be harvested more easily in large quantity, here it is aimed to use ASCs as an alternative cell source. However, H19 activation alone using the Split dCas12a system in ASCs failed to elicit evident chondrogenesis. Therefore, split dCas12a activators are designed more to co-activate other chondroinductive transcription factors (Sox5, Sox6, and Sox9) to synergistically potentiate differentiation. It is found that co-activation of H19/Sox5/Sox6 in ASCs elicited more potent chondrogenic differentiation than activation of Sox5/Sox6/Sox9 or H19 alone. Co-activating H19/Sox5/Sox6 in ASCs significantly augmented in vitro cartilage formation and in vivo calvarial bone healing. These data altogether implicated the potentials of the Split dCas12a system to trigger multiplexed gene activation in ASCs for differentiation pathway reprogramming and tissue regeneration.

The potential role of mechanotransduction in the management of pediatric calvarial bone flap repair.

Pediatric patients suffering traumatic brain injuries may require a decompressive craniectomy to accommodate brain swelling by removing a portion of the skull. Once the brain swelling subsides, the preserved calvarial bone flap is ideally replaced as an autograft during a cranioplasty to restore protection of the brain, as it can reintegrate and grow with the patient during immature skeletal development. However, pediatric patients exhibit a high prevalence of calvarial bone flap resorption post-cranioplasty, causing functional and cosmetic morbidity. This review examines possible solutions for mitigating pediatric calvarial bone flap resorption by delineating methods of stimulating mechanosensitive cell populations with mechanical forces. Mechanotransduction plays a critical role in three main cell types involved with calvarial bone repair, including mesenchymal stem cells, osteoblasts, and dural cells, through mechanisms that could be exploited to promote osteogenesis. In particular, physiologically relevant mechanical forces, including substrate deformation, external forces, and ultrasound, can be used as tools to stimulate bone repair in both in vitro and in vivo systems. Ultimately, combating pediatric calvarial flap resorption may require a combinatorial approach using both cell therapy and bioengineering strategies.

Differential regulation of skeletal stem/progenitor cells in distinct skeletal compartments.

Skeletal stem/progenitor cells (SSPCs), characterized by self-renewal and multipotency, are essential for skeletal development, bone remodeling, and bone repair. These cells have traditionally been known to reside within the bone marrow, but recent studies have identified the presence of distinct SSPC populations in other skeletal compartments such as the growth plate, periosteum, and calvarial sutures. Differences in the cellular and matrix environment of distinct SSPC populations are believed to regulate their stemness and to direct their roles at different stages of development, homeostasis, and regeneration; differences in embryonic origin and adjacent tissue structures also affect SSPC regulation. As these SSPC niches are dynamic and highly specialized, changes under stress conditions and with aging can alter the cellular composition and molecular mechanisms in place, contributing to the dysregulation of local SSPCs and their activity in bone regeneration. Therefore, a better understanding of the different regulatory mechanisms for the distinct SSPCs in each skeletal compartment, and in different conditions, could provide answers to the existing knowledge gap and the impetus for realizing their potential in this biological and medical space. Here, we summarize the current scientific advances made in the study of the differential regulation pathways for distinct SSPCs in different bone compartments. We also discuss the physical, biological, and molecular factors that affect each skeletal compartment niche. Lastly, we look into how aging influences the regenerative capacity of SSPCs. Understanding these regulatory differences can open new avenues for the discovery of novel treatment approaches for calvarial or long bone repair.

The Impact of Genes and the Environment on Calvarial Bone Repair and Remodeling in a Murine Model of Craniosynostosis

INTRODUCTION: TWIST1 mutation leads to Seathre-Chotzen syndrome involving coronal craniosynostosis. Recent studies have shown that exposure to a selective serotonin reuptake inhibitor, citalopram, can cause craniosynostosis. The purpose of this study is to investigate the genetic and environmental mechanisms underlying aberrant bone repair in a murine model of craniosynostosis. METHODS: Thirteen Twist1+/– mutant mice and wild-type (WT) mice with or without in utero citalopram exposure were generated (n = 5 per group): WT, Twist1+/–, WT + citalopram (n = 5), and Twist1+/– + citalopram. Mice underwent calvarial defect surgery followed by microCT imaging at 1, 2, 3, and 4 weeks postoperatively, at which point they were sacrificed for histological analysis. RESULTS: All mice were fully ambulatory after surgery, with no perioperative complications. MicroCT analysis demonstrated that all Twist1+/– mice exposed to citalopram had craniosynostosis and exhibited excessive and disorganized bone growth after injury. Some Twist1+/– mice without citalopram treatment also developed craniosynostosis and exhibited a similar excessive bone growth pattern; Twist1+/– mice without suture fusion had bone repair similar to that of WT mice. WT defects remained incompletely healed at 4-weeks postoperatively. In contrast, the Twist1+/– mice with citalopram exposure demonstrated full bone fill of the defect at 4-weeks. Twist1+/– mice without citalopram demonstrated more bone fill than WT, but the defect was not entirely filled. CONCLUSION: These preliminary findings after calvarial defect surgeries improve our understanding of bone repair in craniosynostosis. By investigating the mechanisms of bone dynamics in craniosynostosis, we can better understand and prevent the factors implicated in resynostosis after surgery.

Single-cell RNA sequencing unravels heterogeneity of skeletal progenitors and cell-cell interactions underlying the bone repair process.

IntroductionActivation of skeletal progenitors upon tissue injury and the subsequent cell fate specification are tightly coordinated in the bone repair process. Although known osteoimmunological signaling networks play important roles in the microenvironment of the bone defect sites, the molecular mechanism underlying the bone repair process has not been fully understood.MethodsTo better understand the behavior of the skeletal progenitors and the heterogeneity of the cells during bone repair at the microenvironmental level, we performed a combinatorial analysis consisting of lineage tracing for skeletal progenitors using the Sox9-CreERT2;R26RtdTomato mouse line followed by single-cell RNA sequencing (scRNA-seq) analysis using a mouse model of calvarial bone repair. To identify a therapeutic target for bone regeneration, further computational analysis was performed focusing on the identification of the cell–cell interactions, followed by pharmacological assessments with a critical-size calvarial bone defect mouse model.ResultsLineage tracing analysis showed that skeletal progenitors marked by Sox9 were activated upon bone injury and contributed to bone repair by differentiating into osteoblasts. The scRNA-seq analysis characterized heterogeneous cell populations at the bone defect sites; the computational analysis predicted a bifurcated lineage from skeletal progenitors toward osteogenic and adipogenic lineages. Chemokine C–C motif ligand 9 (Ccl9) was identified as a signaling molecule that regulates bone regeneration in the mouse model, possibly through the regulation of adipogenic differentiation at the bone defect site.ConclusionMultipotential skeletal progenitors and the direction of the cell differentiation were characterized at single cell resolution in a mouse bone repair model. The Ccl9 signaling pathway may be a key factor directing osteogenesis from the progenitors in the model and may be a therapeutic target for bone regeneration.

Endogenous FGF-2 levels impact FGF-2/BMP-2 growth factor delivery dosing in aged murine calvarial bone defects.

Bone repair in elderly mice has been shown to be improved or negatively impacted by supplementing the highly osteogenic bone morphogenetic protein-2 (BMP-2) with fibroblast growth factor-2 (FGF-2). To better predict the outcome of FGF-2 supplementation, we investigated whether endogenous levels of FGF-2 play a role in optimal dosing of FGF-2 for augmenting BMP-2 activity in elderly mice. In vivo calvarial bone defect studies in Fgf2 knockout mice with wildtype controls were conducted with the growth factors delivered in a highly localized manner from a biomimetic calcium phosphate/polyelectrolyte multilayer coating applied to a bone graft substitute. Endogenous FGF-2 levels were measured in old mice versus young and found to decrease with age. Optimal dosing for improving bone defect repair correlated with levels of endogenous FGF-2, with a larger dose of FGF-2 required to have a positive effect on bone healing in the Fgf2 knockout mice. The same dose in wildtype old mice, with higher levels of FGF-2, promoted chondrogenesis and increased osteoclast activity. The results suggest a personalized medicine approach, based on a knowledge of endogenous levels of FGF-2, should guide FGF-2 supplementation in order to avoid provoking excessive bone resorption and cartilage formation, both of which inhibited calvarial bone repair.

Effect of S53P4 bioactive glass and low-level laser therapy on calvarial bone repair in rats submitted to zoledronic acid therapy.

ABSTRACTPurposeTo evaluate the influence of bioactive glass and photobiomodulation therapy (PBMT) in calvarial bone repair process in rats submitted to zoledronic acid therapy.MethodsTwenty-four rats were selected and treated with the dose of 0.035 mg/kg of zoledronic acid every two weeks, totalizing eight weeks, to induce osteonecrosis. After the drug therapy, surgical procedure was performed to create 5-mm diameter parietal bone defects in the calvarial region. The rats were then randomly assigned to groups according to the following treatments: AZC: control group, treated with blood clot; AZBIO: bone defect filled with bioactive glass; AZL: treated with blood clot and submitted to PBMT; and AZBIOL: treated with bioactive glass S53P4 and submitted to PBMT. Tissue samples were collected and submitted to histomorphometric analysis after 14 and 28 days.ResultsAt 14 days, bone neoformation in the AZBIO (52.15 ± 9.77) and AZBIOL (49.77 ± 13.58) groups presented higher values (p ≤ 0.001) compared to the AZC (23.35 ± 10.15) and AZL groups (23.32 ± 8.75). At 28 days, AZBIO (80.24 ± 5.41)still presented significant higher bone recovery values when compared to AZC (59.59 ± 16.92)and AZL (45.25 ± 5.41) groups (p = 0.048). In the 28-day period, the AZBIOL group didn’t show statistically significant difference with the other groups (71.79 ± 29.38).ConclusionsThe bioactive glass is an effective protocol to stimulate bone neoformation in critical defects surgically created in rats with drug induced osteonecrosis, in the studied periods of 14 and 28 days.

Cranial Suture Regeneration Mitigates Skull and Neurocognitive Defects in Craniosynostosis

Craniosynostosis results from premature fusion of the cranial suture(s), which contain mesenchymal stem cells (MSCs) that are crucial for calvarial expansion in coordination with brain growth. Infants with craniosynostosis have skull dysmorphology, increased intracranial pressure, and complications such as neurocognitive impairment that compromise quality of life. Animal models recapitulating these phenotypes are lacking, hampering development of urgently needed innovative therapies. Here, we show that Twist1+/- mice with craniosynostosis have increased intracranial pressure and neurocognitive behavioral abnormalities, recapitulating features of human Saethre-Chotzen syndrome. Using a biodegradable material combined with MSCs, we successfully regenerated a functional cranial suture that corrects skull deformity, normalizes intracranial pressure, and rescues neurocognitive behavior deficits. The regenerated suture creates a niche into which endogenous MSCs migrated, sustaining calvarial bone homeostasis and repair. MSC-based cranial suture regeneration offers a paradigm shift in treatment to reverse skull and neurocognitive abnormalities in this devastating disease.

Simultaneous nano- and microscale structural control of injectable hydrogels via the assembly of nanofibrous protein microparticles for tissue regeneration

Injectable hydrogels are advantageous as tissue regeneration scaffolds, as they can be delivered through a minimally invasive injection and seamlessly integrate with the target tissues. However, an important shortcoming of current injectable hydrogels is the lack of simultaneous control over their micro- and nanoscale structures. In this article, the authors report a strategy for developing injectable hydrogels that integrate a fibrous nanostructure and porous microstructure. The hydrogels are prepared by using novel nanofibrous microparticles as the building blocks. The protein based nanofibrous microparticles, fabricated by a spray freezing technology, can be injected through a syringe-needle system. A cell-compatible photocuring process can be deployed to connect the microparticles and form a mechanically robust hydrogel scaffold. The inter-particle voids combined to form the interconnected micropores and the diameter of the nanofibers (100–300 nm) closely mimics that of the native extracellular matrix. Compared to the non-porous hydrogels and non-fibrous hydrogels, the microparticle annealed nanofibrous (MANF) hydrogels potently enhance the osteogenic-marker expression (ALP, Runx2, OCT and BSP) and mineralization of human mesenchymal stem cells in vitro. MANF hydrogels also facilitate cell infiltration and enhance neovasculization in a subcutaneous implantation model in vivo. The capacity of MANF hydrogels to promote bone regeneration is investigated in a calvarial bone repair model. MANF hydrogels demonstrate significant higher bone regeneration after 8 weeks, indicating the significant role of microporosity and nanofibrous architecture in bone regeneration.

Collagen-infilled 3D printed scaffolds loaded with miR-148b-transfected bone marrow stem cells improve calvarial bone regeneration in rats.

Differentiation of progenitors in a controlled environment improves the repair of critical-sized calvarial bone defects; however, integrating micro RNA (miRNA) therapy with 3D printed scaffolds still remains a challenge for craniofacial reconstruction. In this study, we aimed to engineer three-dimensional (3D) printed hybrid scaffolds as a new ex situ miR-148b expressing delivery system for osteogenic induction of rat bone marrow stem cells (rBMSCs) in vitro, and also in vivo in critical-sized rat calvarial defects. miR-148b-transfected rBMSCs underwent early differentiation in collagen-infilled 3D printed hybrid scaffolds, expressing significant levels of osteogenic markers compared to non-transfected rBMSCs, as confirmed by gene expression and immunohistochemical staining. Furthermore, after eight weeks of implantation, micro-computed tomography, histology and immunohistochemical staining results indicated that scaffolds loaded with miR-148b-transfected rBMSCs improved bone regeneration considerably compared to the scaffolds loaded with non-transfected rBMSCs and facilitated near-complete repair of critical-sized calvarial defects. In conclusion, our results demonstrate that collagen-infilled 3D printed scaffolds serve as an effective system for miRNA transfected progenitor cells, which has a promising potential for stimulating osteogenesis and calvarial bone repair.

Reconstruction of calvarial bone defects using poly(amino acid)/hydroxyapatite/calcium sulfate composite

The aim of this study was to evaluate the thermal properties and in vivo interface performance of poly(amino acid) (PAA) and a composite containing PAA, hydroxyapatite (HA), and calcium sulfate (CS), with respect to their suitability for skull repair. Biocompatibility was evaluated by implantation of materials into muscles of rabbits for eight weeks. Skull repair was assessed by implanting PAA, the compact PAA/HA/CS composite (c-PAA/HA/CS), and a one-side-porous PAA/HA/CS composite (p-PAA/HA/CS) into rabbit calvarial defects. The results showed that the PAA/HA/CS composite possessed good heat resistance and possessed excellent biocompatibility and osteoconductivity. Guided bone regeneration and calvarial repair were observed, with excellent integration between calvarial tissue and implant. The p-PAA/HA/CS composite performed best in terms of stability and bone bonding between implant and host bone tissue. Thus, the present work provides new information for the potential use of osteoconductive PAA/HA/CS composites with a macrostructure in calvarial bone repair.

BMP-IHH-mediated interplay between mesenchymal stem cells and osteoclasts supports calvarial bone homeostasis and repair

Calvarial bones are connected by fibrous sutures. These sutures provide a niche environment that includes mesenchymal stem cells (MSCs), osteoblasts, and osteoclasts, which help maintain calvarial bone homeostasis and repair. Abnormal function of osteogenic cells or diminished MSCs within the cranial suture can lead to skull defects, such as craniosynostosis. Despite the important function of each of these cell types within the cranial suture, we have limited knowledge about the role that crosstalk between them may play in regulating calvarial bone homeostasis and injury repair. Here we show that suture MSCs give rise to osteoprogenitors that show active bone morphogenetic protein (BMP) signalling and depend on BMP-mediated Indian hedgehog (IHH) signalling to balance osteogenesis and osteoclastogenesis activity. IHH signalling and receptor activator of nuclear factor kappa-Β ligand (RANKL) may function synergistically to promote the differentiation and resorption activity of osteoclasts. Loss of Bmpr1a in MSCs leads to downregulation of hedgehog (Hh) signalling and diminished cranial sutures. Significantly, activation of Hh signalling partially restores suture morphology in Bmpr1a mutant mice, suggesting the functional importance of BMP-mediated Hh signalling in regulating suture tissue homeostasis. Furthermore, there is an increased number of CD200+ cells in Bmpr1a mutant mice, which may also contribute to the inhibited osteoclast activity in the sutures of mutant mice. Finally, suture MSCs require BMP-mediated Hh signalling during the repair of calvarial bone defects after injury. Collectively, our studies reveal the molecular and cellular mechanisms governing cell–cell interactions within the cranial suture that regulate calvarial bone homeostasis and repair.

Cell Type Influences Local Delivery of Biomolecules from a Bioinspired Apatite Drug Delivery System.

Recently, the benefit of step-wise sequential delivery of fibroblast growth factor-2 (FGF-2) and bone morphogenetic protein-2 from a bioinspired apatite drug delivery system on mouse calvarial bone repair was demonstrated. The thicknesses of the nanostructured poly-l-Lysine/poly-l-Glutamic acid polyelectrolyte multilayer (PEM) and the bone-like apatite barrier layer that make up the delivery system, were varied. The effects of the structural variations of the coating on the kinetics of cell access to a cytotoxic factor delivered by the layered structure were evaluated. FGF-2 was adsorbed into the outer PEM, and cytotoxic antimycin-A (AntiA) was adsorbed to the substrate below the barrier layer to detect the timing of the cell access. While MC3T3-E1 osteoprogenitor cells accessed AntiA after three days, the RAW 264.7 macrophage access occurred within 4 h, unless the PEM layer was removed, in which case the results were reversed. Pits were created in the coating by the RAW 264.7 macrophages and initiated delivery, while the osteoprogenitor cell access to drugs occurred through a solution-mediated coating dissolution, at junctions between the islands of crystals. Macrophage-mediated degradation is therefore a mechanism that controls drug release from coatings containing bioinspired apatite.

Systemic Injection of Substance P Promotes Murine Calvarial Repair Through Mobilizing Endogenous Mesenchymal Stem Cells

Craniofacial defect is a critical problem in dental clinic, which has a tremendous impact on patients’ quality of life. Mesenchymal stem cell-based therapy has emerged as a promising approach for tissue defect repair. However, reduced survival after mesenchymal stem cells (MSCs) transplantation remains as a major problem in this area, which hampers the outcome of regeneration. Recently, the mechanism to mobilize endogenous MSCs for tissue regeneration has received increasing attentions, as it does not require exogenous cell transplantation. The primary goal of this study was to confirm the role of intravenous substance P in mobilizing endogenous CD45−CD11b−CD29+ MSCs in critical-sized bone defect animals and to investigate the effects of substance P on calvarial bone repair. Flow cytometry analyses revealed that intravenous substance P promoted the mobilization of endogenous CD45−CD11b−CD29+ MSCs after bone defect. In addition, Micro-CT showed that intravenous substance P improved the outcomes of calvarial bone repair. Furthermore, we discovered that systemic injection of substance P attenuated inflammation and enhanced the survival of the local-transplanted GFP+ MSCs. Our findings suggested that substance P together with its mobilized CD45−CD11b−CD29+ MSCs helped improve calvarial defect repair through regulating inflammatory conditions and promoting the survival of local-transplanted cells.

Calvarial Suture-Derived Stem Cells and Their Contribution to Cranial Bone Repair.

In addition to the natural turnover during life, the bones in the skeleton possess the ability to self-repair in response to injury or disease-related bone loss. Based on studies of bone defect models, both processes are largely supported by resident stem cells. In the long bones, the source of skeletal stem cells has been widely investigated over the years, where the major stem cell population is thought to reside in the perivascular niche of the bone marrow. In contrast, we have very limited knowledge about the stem cells contributing to the repair of calvarial bones. In fact, until recently, the presence of specific stem cells in adult craniofacial bones was uncertain. These flat bones are mainly formed via intramembranous rather than endochondral ossification and thus contain minimal bone marrow space. It has been previously proposed that the overlying periosteum and underlying dura mater provide osteoprogenitors for calvarial bone repair. Nonetheless, recent studies have identified a major stem cell population within the suture mesenchyme with multiple differentiation abilities and intrinsic reparative potential. Here we provide an updated review of calvarial stem cells and potential mechanisms of regulation in the context of skull injury repair.

Simultaneous delivery of hydrophobic small molecules and siRNA using Sterosomes to direct mesenchymal stem cell differentiation for bone repair.

Application of phenamil, a small molecular bone morphogenetic protein (BMP) stimulator, combined with suppression of natural BMP antagonists such as noggin is a promising therapeutic strategy to enhance bone regeneration. Here, we present a novel strategy to co-deliver hydrophobic small molecule phenamil and noggin-targeted siRNA via cationic Sterosomes formed with stearylamine (SA) and high content of cholesterol (Chol) to enhance osteogenesis and bone repair. SA/Chol Sterosomes demonstrated high phenamil encapsulation efficiency, supported sustained release of encapsulated drugs, and significantly reduced drug dose requirements to induce osteogenic differentiation of mesenchymal stem cells (MSCs). Simultaneous deliver of phenamil and noggin siRNA in a single vehicle synergistically enhanced MSC osteogenesis and calvarial bone repair. This study suggests a new non-phospholipid liposomal formulation to simultaneously deliver small molecules and therapeutic genes for combined treatment.

Enhanced critical-size calvarial bone healing by ASCs engineered with Cre/loxP-based hybrid baculovirus.

Calvarial bone repair remains challenging for adults. Although adipose-derived stem cells (ASCs) hold promise to heal bone defects, use of ASCs for critical-size calvarial bone repair is ineffective. Stromal cell-derived factor 1 (SDF-1) is a chemokine capable of triggering stem cell migration. Although recombinant SDF-1 protein is co-delivered with other molecules including BMP-2 to facilitate calvarial bone repair, these approaches did not yield satisfactory healing. This study aimed to exploit a newly developed Cre/loxP-based hybrid baculovirus for efficient gene delivery and prolonged transgene expression in ASCs. We demonstrated that transduction of rat ASCs with the hybrid Cre/loxP-based baculovirus enabled robust and sustained expression of functional BMP-2 and SDF-1. Expression of BMP-2 or SDF-1 alone failed to effectively induce rat ASCs osteogenesis and healing of critical-size calvarial bone defects. Nonetheless, prolonged BMP-2/SDF-1 co-expression in ASCs synergistically activated both Smad and ERK1/2 pathways and hence potentiated the osteogenesis. Consequently, transplantation of the hybrid baculovirus-engineered, BMP-2/SDF-1-expressing ASCs/scaffold constructs potently healed the critical-size (6mm) calvarial bone defects (filling ≈70% of defect volume), which considerably outperformed the calvarial bone repair using BMP-2/SDF-1 delivered with biomaterial-based scaffolds. These data implicated the potential of Cre/loxP-based hybrid baculovirus vector for ASCs engineering and calvarial bone healing.

Improved calvarial bone repair by hASCs engineered with Cre/loxP-based baculovirus conferring prolonged BMP-2 and MiR-148b co-expression.

Repairing large calvarial bone defects remains a challenging task. Previously, it was discovered that that miR-148b, when acting in concert with bone morphogenetic protein 2 (BMP-2), enhanced the osteogenesis of human adipose-derived stem cells (hASCs) and improved calvarial bone healing in nude mice. However, the molecular target of miR-148b remained elusive. Here it is revealed that miR-148b directly targets NOG, whose gene product (noggin) is an antagonist to BMPs and negatively regulates BMP-induced osteogenic differentiation and bone formation. A new Cre/loxP-based baculovirus system was employed to drive prolonged BMP-2 and miR-148b overexpression in hASCs, wherein the BMP-2 overexpression induced noggin expression but the concurrent miR-148b expression downregulated noggin, thus relieving the negative regulatory loop and ameliorating hASC osteogenesis without hindering hASC proliferation or triggering appreciable cytotoxicity. Implantation of the engineered hASCs coexpressing BMP-2 and miR-148b into nude mice enabled substantial repair of critical-size calvarial bone defects (4mm diameter) at 12weeks post-transplantation, filling 83% of the defect area, 75% of bone volume and restoring the bone density to 89% of the original bone density. Such superior healing effects indicate the potential of the Cre/loxP-based baculovirus-mediated BMP-2/miR-148b expression for calvarial bone repair. Copyright © 2016 John Wiley & Sons, Ltd.