STMP and PVPA as Templating Analogs of Noncollagenous Proteins Induce Intrafibrillar Mineralization of Type I Collagen via PCCP Process.

The phosphorylated noncollagenous proteins (NCPs) play a vital role in manipulating biomineralization, while the mechanism of phosphorylation of NCPs in intrafibrillar mineralization of collagen fibril has not been completely deciphered. Poly(vinylphosphonic acid) (PVPA) and sodium trimetaphosphate (STMP) as templating analogs of NCPs induce hierarchical mineralization in cooperation with indispensable sequestration analogs such as polyacrylic acid (PAA) via polymer-induced liquid-like precursor (PILP) process. Herein, STMP-Ca and PVPA-Ca complexes are proposed to achieve rapid intrafibrillar mineralization through polyelectrolyte-Ca complexes pre-precursor (PCCP) process. This strategy is further verified effectively for remineralization of demineralized dentin matrix both in vitro and in vivo. Although STMP micromolecule fails to stabilize amorphous calcium phosphate (ACP) precursor, STMP-Ca complexes facilely permeate into intrafibrillar interstices and trigger phase transition of ACP to hydroxyapatite within collagen. In contrast, PVPA-stabilized ACP precursors lack liquid-like characteristic and crystallize outside collagen due to rigid conformation of PVPA macromolecule, while PVPA-Ca complexes infiltrate into partial intrafibrillar intervals under electrostatic attraction and osmotic pressure as evidenced by intuitionistic 3D stochastic optical reconstruction microscopy (3D-STORM). The study not only extends the variety and size range of polyelectrolyte for PCCP process but also sheds light on the role of phosphorylation for NCPs in biomineralization.

Fibrocyte: A missing piece in the pathogenesis of fibrous epulis.

OBJECTIVES: To explore the role of fibrocytes in the recurrence and calcification of fibrous epulides. METHODS: Different subtypes of fibrous epulides and normal gingival tissue specimens were first collected for histological and immunofluorescence analyses to see if fibrocytes were present and whether they differentiated into myofibroblasts and osteoblasts upon stimulated by transforming growth factor-ß1 (TGF-ß1). Electron microscopy and elemental analysis were used to characterize the extracellular microenvironment in different subtypes of fibrous epulides. Human peripheral blood mononuclear cells (PBMCs) were subsequently isolated from in vitro models to mimic the microenvironment in fibrous epulides to identify whether TGF-ß1 as well as the calcium and phosphorus ion concentration in the extracellular matrix (ECM) of a fibrous epulis trigger fibrocyte differentiation. RESULTS: Fibrous epulides contain fibrocytes that accumulate in the local inflammatory environment and have the ability to differentiate into myofibroblasts or osteoblasts. TGF-ß1 promotes fibrocytes differentiation into myofibroblasts in a concentration-dependent manner, while TGF-ß1 stimulates the fibrocytes to differentiate into osteoblasts when combined with a high calcium and phosphorus environment. CONCLUSIONS: Our study revealed fibrocytes play an important role in the fibrogenesis and osteogenesis in fibrous epulis, and might serve as a therapeutic target for the inhibition of recurrence of fibrous epulides.

In-Depth Occlusion of Dentinal Tubules and Rapid Remineralization of Demineralized Dentin Induced by Polyelectrolyte-Calcium Complexes.

Dentin hypersensitivity (DH) is triggered by external stimuli irking fluid flow through exposed dentinal tubules (DTs). Three commercially available desensitizing agents as control in this study only achieve limited occlusion depths of ≈10 µm in the DTs as well as scarce remineralization of demineralized dentin matrix. Herein, polyelectrolyte-calcium complexes pre-precursor (PCCP) process is proposed for managing DH that demineralized dentin with exposed DTs is rubbed with ultrahighly concentrated polyelectrolyte-calcium suspension (4 g L-1 -5.44 m) followed by phosphate solution (3.25 m), each 10 min, leading to heavy remineralization of demineralized dentin and compact occlusion of the DTs over 200 µm after 1 day of in vitro and in vivo incubation. For the first time, it is demonstrated that the PCCP process relies on the pH-dependent electrostatic attraction between electropositive polyelectrolyte-calcium complexes and electronegative inwalls of DTs comprised of collagen fibrils and hydroxyapatite crystals under alkaline condition. The PCCP process might shed light on a promising dentin desensitizing strategy for DH management via rapid in-depth DT occlusion and remineralization of demineralized dentin.

Regulation of biomineralization by proteoglycans: From mechanisms to application.

Proteoglycans consist of core proteins and one or more covalently-linked glycosaminoglycan chains. They are structurally complex and heterogeneous. Proteoglycans bind to cell surface receptors, cytokines, growth factors and have strong affinity for collagen fibrils. Together with their complex spatial structures and different charge densities, proteoglycans are directly or indirectly involved in biomineralization. The present review focused on the potential mechanisms of proteoglycans-mediated biomineralization. Topics covered include the ability of proteoglycans to influence the proliferation and differentiation of odontoblasts and osteoblasts through complex signaling pathways, as well as regulate the aggregation of collagen fibrils and mineral deposition. The functions of proteoglycans in mineralization regulation and biomimetic properties render them important components in bone tissue engineering. Hence, the integrated impact of proteoglycans on bone formation was also succinctly deliberated. The potential of proteoglycans to function therapeutic targets for relieving the symptoms of ectopic mineralization and mineralization defects was also comprehensively addressed.

Autophagic LC3+ calcified extracellular vesicles initiate cartilage calcification in osteoarthritis.

Pathological cartilage calcification plays an important role in osteoarthritis progression but in which the origin of calcified extracellular vesicles (EVs) and their effects remain unknown. Here, we demonstrate that pathological cartilage calcification occurs in the early stage of the osteoarthritis in which the calcified EVs are closely involved. Autophagosomes carrying the minerals are released in EVs, and calcification is induced by those autophagy-regulated calcified EVs. Autophagy-derived microtubule-associated proteins 1A/1B light chain 3B (LC3)-positive EVs are the major population of calcified EVs that initiate pathological calcification. Release of LC3-positive calcified EVs is caused by blockage of the autophagy flux resulted from histone deacetylase 6 (HDAC6)-mediated microtubule destabilization. Inhibition of HDAC6 activity blocks the release of the LC3-positive calcified EVs by chondrocytes and effectively reverses the pathological calcification and degradation of cartilage. The present work discovers that calcified EVs derived from autophagosomes initiate pathological cartilage calcification in osteoarthritis, with potential therapeutic targeting implication.

Smart, Biomimetic Periosteum Created from the Cerium(III, IV) Oxide-Mineralized Eggshell Membrane.

The periosteum orchestrates the microenvironment of bone regeneration, including facilitating local neuro-vascularization and regulating immune responses. To mimic the role of natural periosteum for bone repair enhancement, we adopted the principle of biomimetic mineralization to delicately inlay amorphous cerium oxide within eggshell membranes (ESMs) for the first time. Cerium from cerium oxide possesses unique ability to switch its oxidation state from cerium III to cerium IV and vice versa, which provides itself promising potential for biomedical applications. ESMs are mineralized with cerium(III, IV) oxide and examined for their biocompatibility. Apart from serving as physical barriers, periosteum-like cerium(III, IV) oxide-mineralized ESMs are biocompatible and can actively regulate immune responses and facilitate local neuro-vascularization along with early-stage bone regeneration in a murine cranial defect model. During the healing process, cerium-inlayed biomimetic periosteum can boost early osteoclastic differentiation of macrophage lineage cells, which may be the dominant mediator of the local repair microenvironment. The present work provides novel insights into expanding the definition and function of a biomimetic periosteum to boost early-stage bone repair and optimize long-term repair with robust neuro-vascularization. This new treatment strategy which employs multifunctional bone-and-periosteum-mimicking systems creates a highly concerted microenvironment to expedite bone regeneration.

Multifunctional Nanomachinery for Enhancement of Bone Healing.

The visionary idea that RNA adopts nonbiological roles in today's nanomaterial world has been nothing short of phenomenal. These RNA molecules have ample chemical functionality and self-assemble to form distinct nanostructures in response to external stimuli. They may be combined with inorganic materials to produce nanomachines that carry cargo to a target site in a controlled manner and respond dynamically to environmental changes. Comparable to biological cells, programmed RNA nanomachines have the potential to replicate bone healing in vitro. Here, an RNA-biomineral nanomachine is developed, which accomplishes intrafibrillar and extrafibrillar mineralization of collagen scaffolds to mimic bone formation in vitro. Molecular dynamics simulation indicates that noncovalent hydrogen bonding provides the energy source that initiates self-assembly of these nanomachines. Incorporation of the RNA-biomineral nanomachines into collagen scaffolds in vivo creates an osteoinductive microenvironment within a bone defect that is conducive to rapid biomineralization and osteogenesis. Addition of RNA-degrading enzymes into RNA-biomineral nanomachines further creates a stop signal that inhibits unwarranted bone formation in tissues. The potential of RNA in building functional nanostructures has been underestimated in the past. The concept of RNA-biomineral nanomachines participating in physiological processes may transform the nanoscopic world of life science.

Extracellular DNA: A Missing Link in the Pathogenesis of Ectopic Mineralization.

Although deoxyribonucleic acid (DNA) is the genetic coding for the very essence of life, these macromolecules or components thereof are not necessarily lost after a cell dies. There appears to be a link between extracellular DNA and biomineralization. Here the authors demonstrate that extracellular DNA functions as an initiator of collagen intrafibrillar mineralization. This is confirmed with in vitro and in vivo biological mineralization models. Because of their polyanionic property, extracellular DNA molecules are capable of stabilizing supersaturated calcium phosphate solution and mineralizing 2D and 3D collagen matrices completely as early as 24 h. The effectiveness of extracellular DNA in biomineralization of collagen is attributed to the relatively stable formation of amorphous liquid droplets triggered by attraction of DNA to the collagen fibrils via hydrogen bonding. These findings suggest that extracellular DNA is biomimetically significant for fabricating inorganic-organic hybrid materials for tissue engineering. DNA-induced collagen intrafibrillar mineralization provides a clue to the pathogenesis of ectopic mineralization in different body tissues. The use of DNase for targeting extracellular DNA at destined tissue sites provides a potential solution for treatment of diseases associated with ectopic mineralization.

Upregulation of mitochondrial dynamics is responsible for osteogenic differentiation of mesenchymal stem cells cultured on self-mineralized collagen membranes.

Collagen membranes crosslinked with high molecular weight polyacrylic acid (HPAA) are capable of self-mineralization via in situ intrafibrillar mineralization. These HPAA-crosslinked collagen membranes (HCM) have been shown to promote osteogenic differentiation of mesenchymal stem cells (MSCs) and enhance bone regeneration in vivo. Nevertheless, the biological triggers involved in those processes and the associated mechanisms are not known. Here, we identified the contribution of mitochondrial dynamics in HCM-mediated osteogenic differentiation of MSCs. Mitochondriogenesis markers were significantly upregulated when MSCs were cultured on HCM, committing the MSCs to osteogenic differentiation. The mitochondria fused to form an interconnected mitochondrial network in response to the high energy requirements. Mitochondrial fission in MSCs was also triggered by HCM; fission slightly declined at 14 days to restore the equilibrium in mitochondrial dynamics. Mitophagy, another event that regulates mitochondrial dynamics, occurred actively to remove dysfunctioned mitochondria and isolate damaged mitochondria from the rest of network. The mitophagy level of MSCs was significantly elevated in the presence of HCM. Taken together, the present findings indicate that upregulation of mitochondrial dynamics via mitochondriogenesis, fusion, fission and mitophagy is responsible for HCM-mediated osteogenic differentiation of MSCs. STATEMENT OF SIGNIFICANCE: High molecular weight polyacrylic acid (HPAA)-crosslinked collagen membrane (HCM) was found to promote in-situ bone regeneration because of it can stimulate osteogenic differentiation of mesenchymal stem cells (MSCs). Nevertheless, the biological triggers involved in those processes and associated mechanisms are not known. This study identifies that activation of mitochondrial dynamics is centrally involved in HCM-mediated osteogenic differentiation of MSCs. The HCM accelerates mitochondriogenesis and regulates homeostasis of the mitochondrial network in response to the increased energy demand for osteogenic differentiation. Concomitantly, mitophagy actively occurs to remove dysfunctioned mitochondria from the rest of the mitochondrial network. Identification of the involvement of mitophagy in HCM-mediated osteogenic differentiation of MSCs opens new vistas in the application of biomimetic mineralization in bone tissue regeneration.

Crosstalk between Bone and Nerves within Bone.

For the past two decades, the function of intrabony nerves on bone has been a subject of intense research, while the function of bone on intrabony nerves is still hidden in the corner. In the present review, the possible crosstalk between bone and intrabony peripheral nerves will be comprehensively analyzed. Peripheral nerves participate in bone development and repair via a host of signals generated through the secretion of neurotransmitters, neuropeptides, axon guidance factors and neurotrophins, with additional contribution from nerve-resident cells. In return, bone contributes to this microenvironmental rendezvous by housing the nerves within its internal milieu to provide mechanical support and a protective shelf. A large ensemble of chemical, mechanical, and electrical cues works in harmony with bone marrow stromal cells in the regulation of intrabony nerves. The crosstalk between bone and nerves is not limited to the physiological state, but also involved in various bone diseases including osteoporosis, osteoarthritis, heterotopic ossification, psychological stress-related bone abnormalities, and bone related tumors. This crosstalk may be harnessed in the design of tissue engineering scaffolds for repair of bone defects or be targeted for treatment of diseases related to bone and peripheral nerves.

Matrix stiffening by self-mineralizable guided bone regeneration.

Collagen membranes produced in vitro with different degrees of intrafibrillar mineralization are potentially useful for guided bone regeneration (GBR). However, highly-mineralized collagen membranes are brittle and difficult for clinical manipulation. The present study aimed at developing an intrafibrillar self-mineralization strategy for GBR membrane by covalently conjugating high-molecular weight polyacrylic acid (HPAA) on Bio-Gide® membranes (BG). The properties of the self-mineralizable membranes (HBG) and their potential to induce bone regeneration were investigated. The HBG underwent the progressive intrafibrillar mineralization as well as the increase in stiffness after immersed in supersaturated calcium phosphate solution, osteogenic medium, or after being implanted into a murine calvarial bone defect. The HBG promoted in-situ bone regeneration via stimulating osteogenic differentiation of mesenchymal stromal cells (MSCs). Hippo signaling was inhibited when MSCs were cultured on the self-mineralized HBG, and in HBG-promoted MSC osteogenesis during in-situ bone regeneration. This resulted in translocation of the transcription co-activators Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ) into the nucleus to induce transcription of genes promoting osteogenic differentiation of MSCs. Taken together, these findings indicated that HBG possessed the ability to self-mineralize in situ via intrafibrillar mineralization. The increase in stiffness of the extracellular matrix expedited in-situ bone regeneration by inactivating the Hippo-YAP/TAZ signaling cascade. STATEMENT OF SIGNIFICANCE: Guided bone regeneration (GBR) membranes made of naturally derived collagen have been widely used in the bone defect restoration. However, application of collagen GBR membranes run into the bottleneck with the challenges like insufficient stress strength, relatively poor dimensional stability and unsatisfactory osteoinductivity. This study develops a modified GBR membrane that can undergo progressive self-mineralization and matrix stiffening in situ. Increase in extracellular matrix stiffness provides the mechanical cues required for MSCs differentiation and expedites in-situ bone regeneration by inactivating the Hippo-YAP/TAZ signaling cascade.

Microbe-Mediated Extracellular and Intracellular Mineralization: Environmental, Industrial, and Biotechnological Applications.

Microbe-mediated mineralization is ubiquitous in nature, involving bacteria, fungi, viruses, and algae. These mineralization processes comprise calcification, silicification, and iron mineralization. The mechanisms for mineral formation include extracellular and intracellular biomineralization. The mineral precipitating capability of microbes is often harnessed for green synthesis of metal nanoparticles, which are relatively less toxic compared with those synthesized through physical or chemical methods. Microbe-mediated mineralization has important applications ranging from pollutant removal and nonreactive carriers, to other industrial and biomedical applications. Herein, the different types of microbe-mediated biomineralization that occur in nature, their mechanisms, as well as their applications are elucidated to create a backdrop for future research.

Establishing an osteoimmunomodulatory coating loaded with aspirin on the surface of titanium primed with phase-transited lysozyme.

BACKGROUND: To improve osseointegration and enhance the success rate of implanted biomaterials, the surface modification technology of bone implants has developed rapidly. Intensive research on osteoimmunomodulation has shown that the surfaces of implants should possess favorable osteoimmunomodulation to facilitate osteogenesis. METHODS: A novel, green and efficient phase-transited lysozyme (PTL) technique was used to prime titanium discs with a positive charge. In addition, sodium hyaluronate (HA) and self-assembled type I collagen containing aspirin (ASA) nanoparticles were decorated on PTL-primed Ti discs via electrostatic interaction. RESULTS: The behaviors of bone marrow stromal cells (BMSCs) on the Ti disc surfaces containing ASA were analyzed in different conditioned media (CM) generated by macrophages. Additionally, the secretion of inflammation-related cytokines of macrophages on the surfaces of different Ti discs was investigated in in vitro experiments, which showed that the Ti surface containing ASA not only supported the migration, proliferation and differentiation of BMSCs but also reduced the inflammatory response of macrophages compared with Ti discs without surface modification. After implantation in vivo, the ASA-modified implant can significantly contribute to bone formation around the implant, which mirrors the evaluation in vitro. CONCLUSION: This study highlights the significant effects of appropriate surface characteristics on the regulation of osteogenesis and osteoimmunomodulation around an implant. Implant modification with ASA potentially provides superior strategies for the surface modification of biomaterials.

MV-mimicking micelles loaded with PEG-serine-ACP nanoparticles to achieve biomimetic intra/extra fibrillar mineralization of collagen in vitro.

Since their discovery, matrix vesicles (MVs) containing minerals have received considerable attention for their role in the mineralization of bone, dentin and calcified cartilage. Additionally, MVs' association with collagen fibrils, which serve as the scaffold for calcification in the organic matrix, has been repeatedly highlighted. The primary purpose of the present study was to establish a MVs-mimicking model (PEG-S-ACP/micelle) in vitro for studying the exact mechanism of MVs-mediated extra/intra fibrillar mineralization of collagen in vivo. In this study, high-concentration serine was used to stabilize the amorphous calcium phosphate (S-ACP), which was subsequently mixed with polyethylene glycol (PEG) to form PEG-S-ACP nanoparticles. The nanoparticles were loaded in the polysorbate 80 micelle through a micelle self-assembly process in an aqueous environment. This MVs-mimicking model is referred to as the PEG-S-ACP/micelle model. By adjusting the pH and surface tension of the PEG-S-ACP/micelle, two forms of minerals (crystalline mineral nodules and ACP nanoparticles) were released to achieve the extrafibrillar and intrafibrillar mineralization, respectively. This in vitro mineralization process reproduced the mineral nodules mediating in vivo extrafibrillar mineralization and provided key insights into a possible mechanism of biomineralization by which in vivo intrafibrillar mineralization could be induced by ACP nanoparticles released from MVs. Also, the PEG-S-ACP/micelle model provides a promising methodology to prepare mineralized collagen scaffolds for repairing bone defects in bone tissue engineering.

Rapid biomimetic remineralization of the demineralized enamel surface using nano-particles of amorphous calcium phosphate guided by chimaeric peptides.

OBJECTIVE: The objective of this study was to develop a rapid and effective method to remineralize human carious-like enamel using chimaeric peptide-mediated nanocomplexes of carboxymethyl chitosan/amorphous calcium phosphate (CMC/ACP), mimicking the mineralizing pattern of the oriented assembly of ACP guided by amelogenin in the biomineralization of enamel. METHODS: CMC/ACP nanocomplex solution was first synthesized through the successive addition of carboxymethyl chitosan, calcium chloride, and dipotassium phosphate into distilled water. ACP nanoparticles were degraded by 1% NaClO from CMC/ACP nanocomplexes. The morphology of the particles at different periods was tested by transmission electron microscopy (TEM). The chimaeric peptides were added to guide the arrangement of ACP nanoparticles and to bind ACP nanoparticles to the demineralized enamel surface specifically. X-ray diffraction (XRD)/scanning electron microscope (SEM)/confocal laser scanning microscopy (CLSM)/nano-indentation tests were applied to check the remineralization effects. RESULTS: CMC/ACP nanocomplexes were obtained and could be kept without precipitation for a long time. After the degradation of NaClO and guidance of chimaeric peptides, ACP nanoparticles were arranged into oriented arrays before transforming into crystals, and the enamel-like crystals were tightly bound onto the demineralized surface. The newly formed enamel-like crystals were nearly well-organized and equipped with strong mechanical properties.