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.

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 stem cells (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.

Maxillary bone augmentation with calvarial bone graft for immediate full-arch rehabilitation: Insights from a 10-year proof-of-concept retrospective analysis.

OBJECTIVE: Evaluate the long-term outcomes of full-arch rehabilitation using immediate dental implant placement and continuous functional loading with full-fixed dental prostheses (FFDPs). MATERIALS AND METHODS: Fifty-six patients received temporary implants (n = 327) at maxillary augmentation with calvarial bone. A provisional acrylic FFDP was immediately loaded onto these implants. After 6 months, the temporary implants were replaced with definitive implants (n = 326) and immediately loaded with a second provisional FFDP (N = 55). Subsequently, a baseline radiograph was taken following a 6-month healing period. The second bridge was then substituted with a definitive FFDP. Primary outcomes included peri-implant marginal bone level (MBL) and definitive implant survival. Secondary outcomes evaluated provisional implant and prostheses survival, complications, and patient satisfaction. RESULTS: The provisional implants had a survival rate of 97.9%. One patient was excluded from further analysis due to loss of temporary implants and first FFDP. The definitive implant survival rate after 10 years was 92.2%, with a moderate but significant decrease in MBL between baseline radiography and 10 years later (-0.08 ± 0.18 vs. -0.24 ± 0.44). However, large individual variations were observed, with 65.8% of implants showing no bone loss and 9.2% showing loss ≥0.5 mm. Sinusitis was experienced by 14.3% of patients upon surgery. Patient satisfaction was high or reported no issues after protocol completion (80%). One patient lost all six definitive implants and definitive FFDP 8.2 years after implant placement. CONCLUSIONS: The described protocol can be regarded as a long-term, highly successful method for full-arch rehabilitation of atrophied maxillae while enabling continuous masticatory and speaking functionality.

Spatial transcriptomics reveals a role for sensory nerves in preserving cranial suture patency through modulation of BMP/TGF-ß signaling.

The patterning and ossification of the mammalian skeleton requires the coordinated actions of both intrinsic bone morphogens and extrinsic neurovascular signals, which function in a temporal and spatial fashion to control mesenchymal progenitor cell (MPC) fate. Here, we show the genetic inhibition of tropomyosin receptor kinase A (TrkA) sensory nerve innervation of the developing cranium results in premature calvarial suture closure, associated with a decrease in suture MPC proliferation and increased mineralization. In vitro, axons from peripheral afferent neurons derived from dorsal root ganglions (DRGs) of wild-type mice induce MPC proliferation in a spatially restricted manner via a soluble factor when cocultured in microfluidic chambers. Comparative spatial transcriptomic analysis of the cranial sutures in vivo confirmed a positive association between sensory axons and proliferative MPCs. SpatialTime analysis across the developing suture revealed regional-specific alterations in bone morphogenetic protein (BMP) and TGF-ß signaling pathway transcripts in response to TrkA inhibition. RNA sequencing of DRG cell bodies, following direct, axonal coculture with MPCs, confirmed the alterations in BMP/TGF-ß signaling pathway transcripts. Among these, the BMP inhibitor follistatin-like 1 (FSTL1) replicated key features of the neural-to-bone influence, including mitogenic and anti-osteogenic effects via the inhibition of BMP/TGF-ß signaling. Taken together, our results demonstrate that sensory nerve-derived signals, including FSTL1, function to coordinate cranial bone patterning by regulating MPC proliferation and differentiation in the suture mesenchyme.

Gene regulatory network from cranial neural crest cells to osteoblast differentiation and calvarial bone development.

Calvarial bone is one of the most complex sequences of developmental events in embryology, featuring a uniquely transient, pluripotent stem cell-like population known as the cranial neural crest (CNC). The skull is formed through intramembranous ossification with distinct tissue lineages (e.g. neural crest derived frontal bone and mesoderm derived parietal bone). Due to CNC's vast cell fate potential, in response to a series of inductive secreted cues including BMP/TGF-ß, Wnt, FGF, Notch, Hedgehog, Hippo and PDGF signaling, CNC enables generations of a diverse spectrum of differentiated cell types in vivo such as osteoblasts and chondrocytes at the craniofacial level. In recent years, since the studies from a genetic mouse model and single-cell sequencing, new discoveries are uncovered upon CNC patterning, differentiation, and the contribution to the development of cranial bones. In this review, we summarized the differences upon the potential gene regulatory network to regulate CNC derived osteogenic potential in mouse and human, and highlighted specific functions of genetic molecules from multiple signaling pathways and the crosstalk, transcription factors and epigenetic factors in orchestrating CNC commitment and differentiation into osteogenic mesenchyme and bone formation. Disorders in gene regulatory network in CNC patterning indicate highly close relevance to clinical birth defects and diseases, providing valuable transgenic mouse models for subsequent discoveries in delineating the underlying molecular mechanisms. We also emphasized the potential regenerative alternative through scientific discoveries from CNC patterning and genetic molecules in interfering with or alleviating clinical disorders or diseases, which will be beneficial for the molecular targets to be integrated for novel therapeutic strategies in the clinic.

Advances and challenges in intravital imaging of craniofacial and dental progenitor cells.

Craniofacial and appendicular bone homeostasis is dynamically regulated by a balance between bone formation and resorption by osteoblasts and osteoclasts, respectively. Despite the developments in multiple imaging techniques in bone biology, there are still technical challenges and limitations in the investigation of spatial/anatomical location of rare stem/progenitor cells and their molecular regulation in tooth and craniofacial bones of living animals. Recent advances in live animal imaging techniques for the craniofacial and dental apparatus can provide new insights in real time into bone stem/progenitor cell dynamics and function in vivo. Here, we review the current inventions and applications of the noninvasive intravital imaging technique and its practical uses and limitations in the analysis of stem/progenitor cells in craniofacial and dental apparatus in vivo. Furthermore, we also explore the potential applications of intravital microscopy in the dental field.

Investigation into the effects of leukemia inhibitory factor on the bone repair capacity of BMSCs-loaded BCP scaffolds in the mouse calvarial bone defect model.

Leukemia inhibitory factor (LIF) is known to play a major role in bone physiology. In the present study, we examined the in vitro effects of LIF on osteoblast differentiation of bone marrow stem cells (BMSCs) and explored in vivo effects of LIF on the bone repair capacity of BMSCs-loaded biphasic calcium phosphate (BCP) scaffolds in mouse calvarial bone defect model. The mRNA and protein expression levels in the BMSCs were determined by quantitative real-time PCR and western blot, respectively; the in vitro osteoblast differentiation of the BMSCs was evaluated by using Alizarin Red S staining. The bone volume and bone density in the repaired calvarial bone defect were determined by Micro-CT. Bone regeneration was also histologically evaluated by hematoxylin and eosin staining and Masson's trichrome staining. Hypoxia treatment induced the up-regulation of Lif mRNA and LIF protein in the BMSCs. Lif overexpression up-regulated the mRNA expression levels of osteopontin and Runt-related transcription factor 2, and increased intensity of Alizarin Red S staining in the BMSCs; while Lif silence exerted the opposite effects. The in vivo studies showed that implantation of Lif-overexpressing BMSCs-loaded BCP scaffolds significantly increased the bone volume and bone density at 4 and 8 weeks after transplantation, and promoted the regeneration of bone tissues in the mouse calvarial bone defect at 8 weeks after transplantation when compared to the BMSCs-loaded BCP scaffolds group; while Lif-silencing BMSCs-loaded BCP scaffolds had the opposite effects. The present study for the first time demonstrated that LIF promoted the in vitro osteoblast differentiation of hypoxia-treated BMSCs; and further studies revealed that LIF exerted enhanced effects on the bone repair capacity of BMSCs-load BCP scaffolds in mouse calvarial bone defect model. However, future studies are warranted to determine the detailed mechanisms of LIF in the large-scale bone defect repair.

Reconstruction of the severely atrophic edentulous maxillae with calvarial bone grafts.

OBJECTIVES: Treatment of the severely atrophic maxilla with dental implants is challenging due to the insufficient horizontal and vertical bone volume and centripetal resorption pattern of the maxilla. Bone-augmentation procedures are often necessary prior to implant placement. The objective of this study was to assess the suitability of using calvarial bone grafts to enable implant placement in severely atrophic maxillae. MATERIAL AND METHODS: Seventeen patients with severe atrophic edentulous maxillae were reconstructed with autogenous calvarial bone grafts. After a 4-month healing period, the patients received a total of 134 dental implants, which were left to heal in submerged positions for 3 months before prosthetic rehabilitation was performed. Patients were followed clinically and radiographically for an average observation period of 53.94 months. RESULTS: At the intraoral recipient sites, two infections developed, causing partial loss of the respective bone grafts. Implant placement, however, was possible at all sites. No donor-site complications occurred. Two of 134 implants were lost in two patients prior to prosthetic loading. The implant survival rate was 98.51%. The implant success rate was 87.6%, and a mean marginal bone loss of 0.62 mm (SD 0.77 mm) was documented. CONCLUSIONS: Patients with severe bone atrophy of the edentulous maxilla can be successfully reconstructed with calvarial bone grafts and dental implants and show a stable clinical and radiographic situation after a mean observation period of 53 months.

Novel bioactivity of phosvitin in connective tissue and bone organogenesis revealed by live calvarial bone organ culture models.

Egg yolk phosvitin is one of the most highly phosphorylated extracellular matrix proteins known in nature with unique physico-chemical properties deemed to be critical during ex-vivo egg embryo development. We have utilized our unique live mouse calvarial bone organ culture models under conditions which dissociates the two bone remodeling stages, viz., resorption by osteoclasts and formation by osteoblasts, to highlight important and to date unknown critical biological functions of egg phosvitin. In our resorption model live bone cultures were grown in the absence of ascorbate and were stimulated by parathyroid hormone (PTH) to undergo rapid osteoclast formation/differentiation with bone resorption. In this resorption model native phosvitin potently inhibited PTH-induced osteoclastic bone resorption with simultaneous new osteoid/bone formation in the absence of ascorbate (vitamin C). These surprising and critical observations were extended using the bone formation model in the absence of ascorbate and in the presence of phosvitin which supported the above results. The results were corroborated by analyses for calcium release or uptake, tartrate-resistant acid phosphatase activity (marker for osteoclasts), alkaline phosphatase activity (marker for osteoblasts), collagen and hydroxyproline composition, and histological and quantitative histomorphometric evaluations. The data revealed that the discovered bioactivity of phosvitin mirrors that of ascorbate during collagen synthesis and the formation of new osteoid/bone. Complementing those studies use of the synthetic collagen peptide analog and cultured calvarial osteoblasts in conjunction with mass spectrometric analysis provided results that augmented the bone organ culture work and confirmed the capacity of phosvitin to stimulate differentiation of osteoblasts, collagen synthesis, hydroxyproline formation, and biomineralization. There are striking implications and interrelationships of this affect that relates to the evolutionary inactivation of the gene of an enzyme L-gulono-γ-lactone oxidase, which is involved in the final step of ascorbate biosynthesis, in many vertebrate species including passeriform birds, reptiles and teleost fish whose egg yolk contain phosvitin. These represent examples of how developing ex-vivo embryos of such species can achieve connective tissue and skeletal system formation in the absence of ascorbate.

FAD104, a regulatory factor of adipogenesis, acts as a novel regulator of calvarial bone formation.

Osteogenesis is a complex process that is orchestrated by several growth factors, extracellular cues, signaling molecules, and transcriptional factors. Understanding the mechanisms of bone formation is pivotal for clarifying the pathogenesis of bone diseases. Previously, we reported that fad104 (factor for adipocyte differentiation 104), a novel positive regulator of adipocyte differentiation, negatively regulated the differentiation of mouse embryonic fibroblasts into osteocytes. However, the physiological role of fad104 in bone formation has not been elucidated. Here, we clarified the role of fad104 in bone formation in vivo and in vitro. fad104 disruption caused craniosynostosis-like premature ossification of the calvarial bone. Furthermore, analyses using primary calvarial cells revealed that fad104 negatively regulated differentiation and BMP/Smad signaling pathway. FAD104 interacted with Smad1/5/8. The N-terminal region of FAD104, which contains a proline-rich motif, was capable of binding to Smad1/5/8. We demonstrated that down-regulation of Smad1/5/8 phosphorylation by FAD104 is dependent on the N-terminal region of FAD104 and that fad104 functions as a novel negative regulator of BMP/Smad signaling and is required for proper development for calvarial bone. These findings will aid a comprehensive description of the mechanism that controls normal and premature calvarial ossification.

Effect of rhBMP-2 dose on bone formation/maturation in a rat critical-size calvarial defect model.

BACKGROUND: Application of recombinant human bone morphogenetic protein-2 (rhBMP-2) has been associated with significant adverse events in craniofacial settings, including swelling and seroma formation. Recent work has demonstrated an inverse relationship between bone formation/maturation and rhBMP-2 dose, frequency/severity of adverse events increasing with rising dose. OBJECTIVE: The objective of this study was to determine the most effective dose for rhBMP-2 soak-loaded onto an absorbable collagen sponge (ACS) carrier for bone formation/maturation using an established defect model. METHODS: One hundred sixty-eight outbred male Sprague-Dawley rats, age 11-13 weeks, weight 325-375 g randomized into seven groups of 24 subdivided into groups of eight, were used to provide radiographic and light microscopy observations of bone formation/maturation and aberrant healing events at 2, 4 and 8 weeks following application of rhBMP-2/ACS into critical-size, ø8-mm, through-through, calvarial osteotomy defects for a dose of 1.25, 2.5, 5.0, 10.0 and 20.0 µg rhBMP-2/defect, or serve as ACS or sham-surgery controls. RESULTS: rhBMP-2 dosages ≥ 2.5 µg/defect showed histological defect closure >90% within 2 weeks, and complete resolution within 4 weeks. Adverse healing events including swelling, excessive bone formation or seroma formation could not be determined with certainty in this defect model. Notably ACS control sites showed complete defect closure at the 8-week healing interval. CONCLUSIONS: rhBMP-2/ACS accelerates local bone formation in the rat critical-size through-through calvarial defect model once reaching an osteoinductive dose threshold. This threshold may already be reached at a 1.25-/2.5-µg dose in this model. No further enhancement to bone formation/maturation may be observed adding rhBMP-2 above the 2.5-µg dose. The 1.25-20.0 µg dose range did not invoke appreciable aberrant healing events.

Piezoelectric technology for pediatric autologous cranioplasty.

Objective : Pediatric patients with skull defects larger than available sources for splitting bicortical bone have limited options for autogenous cortical bone cranioplasty. Piezoelectric instruments allow donor bone to be chosen based on the best possible contour rather than the presence of bicortical bone. We present the use of piezoelectric technology to split thin unicortical calvarium for autogenous cranioplasty in a series of pediatric patients. Design : Retrospective review of a series of pediatric patients requiring reconstruction for skull defects. Patients/Intervention : Our series included a 2-year-old with a parietal skull tumor and resultant 3 × 3-cm defect after craniectomy, a 2-year-old with a 3 × 3-cm defect after excision of an occipital skull tumor, a 10-year-old with a 4 × 5-cm skull defect after excision of an occipital skull tumor, and a 13-year-old who suffered a gunshot to the forehead with a 12 × 7-cm frontal skull defect. We used a piezoelectric saw to precisely and safely split unicortical and bicortical cranium that ranged from 1 to 3 mm in thickness. The inner layer was used to reconstruct the donor site; whereas, the outer layer was used for the craniectomy defect. Conclusion : The piezoelectric saw allows unicortical bone to be split and used for cortical bone cranioplasty. This technology allows choice of donor site based on the best contour rather than the presence of bicortical bone. This technique expands the possibilities of autogenous cranioplasty and enables primary repair of cranial defects that would otherwise require secondary cranioplasty with remote donor sites, foreign materials, or unstable particulate cranioplasty.

Correlation between Ca Release and Osteoconduction by 3D-Printed Hydroxyapatite-Based Templates.

The application of hydroxyapatite (HA)-based templates is quite often seen in bone tissue engineering since that HA is an osteoconductive bioceramic material, which mimics the inorganic component of mineralized tissues. However, the reported osteoconductivity varies in vitro and in vivo, and the levels of calcium (Ca) release most favorable to osteoconduction have yet to be determined. In this study, HA-based templates were fabricated by melt-extrusion 3D-printing and characterized in order to determine a possible correlation between Ca release and osteoconduction. The HA-based templates were blended with poly(lactide-co-trimethylene carbonate) (PLATMC) at three different HA ratios: 10, 30, and 50%. The printability and physical properties of the HA templates were compared with those of pristine PLATMC. In vitro, osteoconductivity was assessed using seeded human bone marrow-derived mesenchymal stem cells. A mild rate of Ca release was observed for HA10 templates, which exhibited higher mineralized extracellular matrix (ECM) secretion than PLATMC at 14 and 21 days. In contrast, the high rate of Ca release exhibited by HA30 and HA50 templates was associated with reduced osteoconduction and impeded mineralized ECM secretion in vitro. Similar results were observed in vivo. In the calvarial defect model in rabbit, PLATMC and HA10 templates exhibited the highest amount of new bone formation, with obvious contact osteogenesis on their surfaces. In contrast, HA30 and HA50 exhibited distant osteogenesis and reduced amounts of new bone ingrowth. It is concluded that HA-based templates are osteoconductive only at low rates of Ca release.

Boron substitution in silicate bioactive glass scaffolds to enhance bone differentiation and regeneration.

Commercially available bioactive glasses (BAGs) are exclusively used in powder form, due to their tendency to crystallize. Silicate BAG 1393 was developed to allow fiber drawing and scaffold sintering, but its slow degradation limits its potential. To enable scaffold manufacturing while maintaining glass dissolution rate close to that of commercially available BAGs, the borosilicate glass 1393B20 was developed. This study investigates the potential of 1393B20 scaffolds to support bone regeneration and mineralization in vitro and in vivo, in comparison to silicate 1393. Both scaffolds supported human adipose stem cells proliferation, either in direct contact for the 1393, or mainly around for the 1393B20. Similarly, both BAGs induced osteogenesis and angiogenesis in vitro, with a better pro-angiogenic influence of the 1393B20. In addition, these scaffolds supported bone regeneration and osteoclast/osteoblast activity in vivo in critical-sized rat calvarial defect. Nevertheless, mineralization and collagen formation were significantly enhanced for the 1393B20, at 3-months post-implantation, assigned to faster and more complete dissolution of the scaffolds. Thus, 1393B20 demonstrates greater promise for bone tissue engineering certainly due to its time-controlled release of boron and silicon. STATEMENT OF SIGNIFICANCE: Bioactive glasses (BAGs) show great promise in bone tissue engineering as they effectively bond with bone tissue, fostering integration and regeneration. Silicate BAG 1393 was developed to allow fiber drawing and scaffold sintering, but its slow degradation limits its potential. To enable scaffold manufacturing while maintaining glass dissolution rate close to that of commercially available BAGs, the borosilicate glass 1393B20 was developed. Both BAGs induced osteogenesis and angiogenesis in vitro, with a better pro-angiogenic influence of the 1393B20. The presence of boron in the 1393B20 enhanced mineralization and collagen formation in vivo compared to 1393, probably due to its faster dissolution rate. Here, 1393B20 demonstrated greater promise for bone tissue engineering compared to the well-known 1393 BAG.

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.

Effect of lyophilized gelatin-norbornene cryogel size on calvarial bone regeneration.

Molding processes with molds containing topographical structures have been used for fabrication of hydrogel and cryogel particles. However, they can involve difficulties in separation of fabricated particles with complex shape from the molds or repeated fabrication of the particles although the overall processes do not require much skill and equipment. In this study, molds with etched superhydrophobic patterns have been developed by etching polytetrafluoroethylene (PTFE) blocks in user-defined designs with a femtosecond (FS) laser-based etching system. Lyophilized cryogel particles with various designs and sizes were fabricated by molding precursors with these PTFE molds. Additionally, the clean and easy separation of particles from the molds allowed repeated fabrication of the particles. For an application, relatively 'big' gelatin-norbornene (GelNB) cryogel particles prepared via molding with polydimethylsiloxane (PDMS) molds, swelling in phosphate buffered saline (PBS) and slicing height in half and 'small' GelNB cryogel particles fabricated with the PTFE molds were fabricated. Then, they were used to study scaffold size effect on calvarial bone regeneration. The molds generated with the FS laser-based etching system can be useful for various applications that require the mass production of cryogel particles in various geometries.

Skull metastases from extramammary Paget's disease emerging 8 years after initial treatment with no local progression: illustrative case.

BACKGROUND: The foci of distant metastasis from extramammary Paget's disease (EMPD) are the lung, liver, truncal bones, vertebrae, and brain. However, skull metastases have not been reported. OBSERVATIONS: The authors treated a patient with calvarial and skull base metastases from EMPD who had undergone wide local resection of EMPD 8 years before, and they report his clinical course. LESSONS: Because EMPD with distant metastasis is fatal, it should be recognized that EMPD can metastasize to the skull even when it seemed to be in remission for several years.

PTH-Induced Bone Regeneration and Vascular Modulation Are Both Dependent on Endothelial Signaling.

The use of a bone allograft presents a promising approach for healing nonunion fractures. We have previously reported that parathyroid hormone (PTH) therapy induced allograft integration while modulating angiogenesis at the allograft proximity. Here, we hypothesize that PTH-induced vascular modulation and the osteogenic effect of PTH are both dependent on endothelial PTH receptor-1 (PTHR1) signaling. To evaluate our hypothesis, we used multiple transgenic mouse lines, and their wild-type counterparts as a control. In addition to endothelial-specific PTHR1 knock-out mice, we used mice in which PTHR1 was engineered to be constitutively active in collagen-1α+ osteoblasts, to assess the effect of PTH signaling activation exclusively in osteoprogenitors. To characterize resident cell recruitment and osteogenic activity, mice in which the Luciferase reporter gene is expressed under the Osteocalcin promoter (Oc-Luc) were used. Mice were implanted with calvarial allografts and treated with either PTH or PBS. A micro-computed tomography-based structural analysis indicated that the induction of bone formation by PTH, as observed in wild-type animals, was not maintained when PTHR1 was removed from endothelial cells. Furthermore, the induction of PTH signaling exclusively in osteoblasts resulted in significantly less bone formation compared to systemic PTH treatment, and significantly less osteogenic activity was measured by bioluminescence imaging of the Oc-Luc mice. Deletion of the endothelial PTHR1 significantly decreased the PTH-induced formation of narrow blood vessels, formerly demonstrated in wild-type mice. However, the exclusive activation of PTH signaling in osteoblasts was sufficient to re-establish the observed PTH effect. Collectively, our results show that endothelial PTHR1 signaling plays a key role in PTH-induced osteogenesis and has implications in angiogenesis.

Effect of chitosan/inorganic nanomaterial scaffolds on bone regeneration and related influencing factors in animal models: A systematic review.

Bone tissue engineering (BTE) provides a promising alternative for transplanting. Due to biocompatibility and biodegradability, chitosan-based scaffolds have been extensively studied. In recent years, many inorganic nanomaterials have been utilized to modify the performance of chitosan-based materials. In order to ascertain the impact of chitosan/inorganic nanomaterial scaffolds on bone regeneration and related key factors, this study presents a systematic comparison of various scaffolds in the calvarial critical-sized defect (CSD) model. A total of four electronic databases were searched without publication date or language restrictions up to April 2022. The Animal Research Reporting of In Vivo Experiments 2.0 guidelines (ARRIVE 2.0) were used to assess the quality of the included studies. Moreover, the risk of bias (RoB) was evaluated via the Systematic Review Center for Laboratory Animal Experimentation (SYRCLE) tool. After the screening, 22 studies were selected. None of these studies achieved high quality or had a low RoB. In the available studies, scaffolds reconstructed bone defects in radically different extensions. Several significant factors were identified, including baseline characteristics, physicochemical properties of scaffolds, surgery details, and scanning or reconstruction parameters of micro-computed tomography (micro-CT). Further studies should focus on not only improving the osteogenic performance of the scaffolds but also increasing the credibility of studies through rigorous experimental design and normative reports.

The use of heparin/polycation coacervate sustain release system to compare the bone regenerative potentials of 5 BMPs using a critical sized calvarial bone defect model.

Nonunion following bone fracture and segmental bone defects are challenging clinical conditions. To combat this clinical dilemma, development of new bone tissue engineering therapies using biocompatible materials to deliver bone growth factors is desirable. This aim of this study is to use a heparin/polycation coacervate sustained-release platform to compare 5 bone morphogenetic proteins (BMPs) for promoting bone defect healing in a critical sized calvarial defect model. The in vitro 3D osteogenic pellet cultures assays demonstrated that BMPs 2, 4, 6, 7 and 9 all enhanced mineralization in vitro compared to the control group. BMP2 resulted in higher mineralized volume than BMP4 and BMP6. All BMPs and the control group activated the pSMAD5 signaling pathway and expressed osterix (OSX). The binding of BMP2 with coacervate significantly increased the coacervate average particle size. BMP2, 4, 6, & 7 bound to coacervate significantly increased the Zeta potential of the coacervate while BMP9 binding showed insignificant increase. Furthermore, using a monolayer culture osteogenic assay, it was found that hMDSCs cultured in the coacervate BMP2 osteogenic medium expressed higher levels of RUNX2, OSX, ALP and COX-2 compared to the control and BMPs 4, 6, 7 & 9. Additionally, the coacervate complex can be loaded with up to 2 µg of BMP proteins for sustained release. In vivo, when BMPs were delivered using the coacervate sustained release system, BMP2 was identified to be the most potent BMP promoting bone regeneration and regenerated 10 times of new bone than BMPs 4, 6 & 9. BMP7 also stimulated robust bone regeneration when compared to BMPs 4, 6 & 9. The quality of the newly regenerated bone by all BMPs delivered by coacervate is equivalent to the host bone consisting of bone matrix and bone marrow with normal bone architecture. Although the defect was not completely healed at 6 weeks, coacervate sustain release BMPs, particularly BMP2 and BMP7, could represent a new strategy for treatment of bone defects and non-unions.