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.     

Synthesis of oligo(poly(ethylene glycol) fumarate)

This protocol describes the synthesis of oligo(poly(ethylene glycol) fumarate) (OPF; 1-35 kDa; a polymer useful for tissue engineering applications) by a one-pot reaction of poly(ethylene glycol) (PEG) and fumaryl chloride. The procedure involves three parts: dichloromethane and PEG are first dried; the reaction step follows, in which fumaryl chloride and triethylamine are added dropwise to a solution of PEG in dichloromethane; and finally, the product solution is filtered to remove by-product salt, and the OPF product is twice crystallized, washed and dried under vacuum. The reaction is affected by the molecular weight of PEG and reactant molar ratio. The OPF product is cross-linked by radical polymerization by either a thermally induced or ultraviolet-induced radical initiator, and the physical properties of the OPF oligomer and resulting cross-linked hydrogel are easily tailored by varying PEG molecular weight. OPF hydrogels are injectable, they polymerize in situ and they undergo biodegradation by hydrolysis of ester bonds. The expected time required to complete this protocol is 6 d.     

Self-healing and injectable hybrid hydrogel for bone regeneration of femoral head necrosis and defect

Background

HA modified by bisphosphonate (BP) (HA-BP) was synthesized by chemical reaction and possessed promising properties such as self-healing, injection ability, and strong adhesion. The main aim of this study was to confirm its role in promoting osteogenic differentiation in vitro and bone regeneration in vivo.

In vitro cytotoxicity of unsaturated oligo[poly(ethylene glycol) fumarate] macromers and their cross-linked hydrogels

Currently, oligo[poly(ethylene glycol) fumarate] (OPF) hydrogels are being investigated as an injectable and biodegradable system for tissue engineering applications. In this study, cytotoxicity of each component of the OPF hydrogel formulation and the resulting cross-linked network was examined. Specifically, OPF synthesized with poly(ethylene glycol) (PEG) of different molecular weights (MW), the cross-linking agent [PEG-diacrylate (PEG-DA)], and the redox initiator pair [ammonium persulfate (APS) and ascorbic acid (AA)] were evaluated for cytotoxicity at 2 and 24 h using marrow stromal cells (MSCs) as model cells. The effect of leachable byproducts of OPF hydrogels on cytotoxicity was also investigated. Upon exposure to various concentrations of OPF for 2 h, greater than 50% of the MSCs were viable, regardless of OPF molecular weight or concentration in the media. After 24 h, the MSCs maintained more than 75% viability except for OPF concentrations higher than 25% (w/v). When examining the cross-linking agent, PEG-DA of higher MW (3400) demonstrated significantly higher viability compared to PEG-DA with MW 575 at all concentrations tested. Considering initiators, when MSCs were exposed to AA and APS, as well as the combination of AA and APS, higher viability was observed at lower concentrations. Once cross-linked, the leachable products from the OPF hydrogels had minimal adverse effects on the viability of MSCs (percentage of live cells was higher than 90% regardless of hydrogel types). The results suggest that, after optimization of cross-linking parameters, OPF-based hydrogels hold promise as novel injectable scaffolds or cell carriers in tissue engineering.     

Injectable biodegradable hydrogels for embryonic stem cell transplantation: improved cardiac remodelling and function of myocardial infarction

In this study, an injectable, biodegradable hydrogel composite of oligo[poly(ethylene glycol) fumarate] (OPF) was investigated as a carrier of mouse embryonic stem cells (mESCs) for the treatment of myocardial infarction (MI). The OPF hydrogels were used to encapsulate mESCs. The cell differentiation in vitro over 14 days was determined via immunohistochemical examination. Then, mESCs encapsulated in OPF hydrogels were injected into the LV wall of a rat MI model. Detailed histological analysis and echocardiography were used to determine the structural and functional consequences after 4 weeks of transplantation. With ascorbic acid induction, mESCs could differentiate into cardiomyocytes and other cell types in all three lineages in the OPF hydrogel. After transplantation, both the 24-hr cell retention and 4-week graft size were significantly greater in the OPF + ESC group than that of the PBS + ESC group (P < 0.01). Four weeks after transplantation, OPF hydrogel alone significantly reduced the infarct size and collagen deposition and improved the cardiac function. The heart function and revascularization improved significantly, while the infarct size and fibrotic area decreased significantly in the OPF + ESC group compared with that of the PBS + ESC, OPF and PBS groups (P < 0.01). All treatments had significantly reduced MMP2 and MMP9 protein levels compared to the PBS control group, and the OPF + ESC group decreased most by Western blotting. Transplanted mESCs expressed cardiovascular markers. This study suggests the potential of a method for heart regeneration involving OPF hydrogels for stem cell encapsulation and transplantation.     

Characterization of photo-cross-linked oligo[poly(ethylene glycol) fumarate] hydrogels for cartilage tissue engineering

Photo-cross-linkable oligo[poly(ethylene glycol) fumarate] (OPF) hydrogels have been developed for use in tissue engineering applications. We demonstrated that compressive modulus of these hydrogels increased with increasing polymer concentration, and hydrogels with different mechanical properties were formed by altering the ratio of cross-linker/polymer in precursor solution. Conversely, swelling of hydrogels decreased with increasing polymer concentration and cross-linker/polymer ratio. These hydrogels are degradable and degradation rates vary with the change in cross-linking level. Chondrocyte attachment was quantified as a method for evaluating adhesion of cells to the hydrogels. These data revealed that cross-linking density affects cell behavior on the hydrogel surfaces. Cell attachment was greater on the samples with increased cross-linking density. Chondrocytes on these samples exhibited spread morphology with distinct actin stress fibers, whereas they maintained their rounded morphology on the samples with lower cross-linking density. Moreover, chondrocytes were photoencapsulated within various hydrogel networks. Our results revealed that cells encapsulated within 2-mm thick OPF hydrogel disks remained viable throughout the 3-week culture period, with no difference in viability across the thickness of hydrogels. Photoencapsulated chondrocytes expressed the mRNA of type II collagen and produced cartilaginous matrix within the hydrogel constructs after three weeks. These findings suggest that photo-cross-linkable OPF hydrogels may be useful for cartilage tissue engineering and cell delivery applications.     

Bone regeneration at the expense of Bioceramic/Hydrogel composite: Clinical evidence

It is often necessary to restore bone structure following the loss of osseous substance. Therefore, the use of bone substitutes made of synthetic calcium phosphate (CaP) ceramics has become more widespread. Granular forms of these biomaterials have demonstrated osteogenic capacities within various sites. Recently, a combination of CaP microporous granules and hydrosoluble polymers was developed (In’Oss?). This composite material is non-self-hardening, injectable, and moldable. The purpose of this non-interventional clinical study was to demonstrate the safety and efficacy of these bioceramic/hydrogel bone substitutes in bone regeneration following maxillofacial surgery. In’Oss? biomaterial was injected into bone defects created by tooth removal in 78 patients. Notably, no infectious or foreign body reactions were reported during at least 17 months of follow up for the study. Regeneration of bone architecture was observed by histomorphometry.     

The size exclusion characteristics of type I collagen: implications for the role of noncollagenous bone constituents in mineralization

The mineral in bone is located primarily within the collagen fibril, and during mineralization the fibril is formed first and then water within the fibril is replaced with mineral. The collagen fibril therefore provides the aqueous compartment in which mineral grows. Although knowledge of the size of molecules that can diffuse into the fibril to affect crystal growth is critical to understanding the mechanism of bone mineralization, there have been as yet no studies on the size exclusion properties of the collagen fibril. To determine the size exclusion characteristics of collagen, we developed a gel filtration-like procedure that uses columns containing collagen from tendon and bone. The elution volumes of test molecules show the volume within the packed column that is accessible to the test molecules, and therefore reveal the size exclusion characteristics of the collagen within the column. These experiments show that molecules smaller than a 6-kDa protein diffuse into all of the water within the collagen fibril, whereas molecules larger than a 40-kDa protein are excluded from this water. These studies provide an insight into the mechanism of bone mineralization. Molecules and apatite crystals smaller than a 6-kDa protein can diffuse into all water within the fibril and so can directly impact mineralization. Although molecules larger than a 40-kDa protein are excluded from the fibril, they can initiate mineralization by forming small apatite crystal nuclei that diffuse into the fibril, or can favor fibril mineralization by inhibiting apatite growth everywhere but within the fibril.     

Biomimetic mineralization of woven bone-like nanocomposites: role of collagen cross-links

Ideal biomaterials for bone grafts must be biocompatible, osteoconductive, osteoinductive and have appropriate mechanical properties. For this, the development of synthetic bone substitutes mimicking natural bone is desirable, but this requires controllable mineralization of the collagen matrix. In this study, densified collagen films (up to 100 μm thick) were fabricated by a plastic compression technique and cross-linked using carbodiimide. Then, collagen-hydroxyapatite composites were prepared by using a polymer-induced liquid-precursor (PILP) mineralization process. Compared to traditional methods that produce only extrafibrillar hydroxyapatite (HA) clusters on the surface of collagen scaffolds, by using the PILP mineralization process, homogeneous intra- and extrafibrillar minerals were achieved on densified collagen films, leading to a similar nanostructure as bone, and a woven microstructure analogous to woven bone. The role of collagen cross-links on mineralization was examined and it was found that the cross-linked collagen films stimulated the mineralization reaction, which in turn enhanced the mechanical properties (hardness and modulus). The highest value of hardness and elastic modulus was 0.7 ± 0.1 and 9.1 ± 1.4 GPa in the dry state, respectively, which is comparable to that of woven bone. In the wet state, the values were much lower (177 ± 31 and 8 ± 3 MPa) due to inherent microporosity in the films, but still comparable to those of woven bone in the same conditions. Mineralization of collagen films with controllable mineral content and good mechanical properties provide a biomimetic route toward the development of bone substitutes for the next generation of biomaterials. This work also provides insight into understanding the role of collagen fibrils on mineralization.     

Elastic deformation of mineralized collagen fibrils: an equivalent inclusion based composite model

Mineralized collagen fibrils are the basic building blocks of bone tissue at the supramolecular level. Several disease states, manipulation of the expression of specific proteins involved in biomineralization, and treatment with different agents alter the extent of mineralization as well as the morphology of mineral crystals which in turn affect the mechanical function of bone tissue. An experimental assessment of mineralized fibers' mechanical properties is challenged by their small size, leaving analytical and computational models as a viable alternative for investigation of the fibril-level mechanical properties. In the current study the variation of the elastic stiffness tensor of mineralized collagen fibrils with changing mineral volume fraction and mineral aspect ratios was predicted via a micromechanical model. The partitioning of applied stresses between mineral and collagen phases is also predicted for normal and shear loading of fibrils. Model predictions resulted in transversely isotropic collagen fibrils in which the modulus along the longer axis of the fibril was the greatest. All the elastic moduli increased with increasing mineral volume fraction whereas Poisson's ratios decreased with the exception of v12 (=v21). The partitioning of applied stresses were such that the stresses acting on mineral crystals were about 1.5, 15, and 3 times greater than collagen stresses when fibrils were loaded transversely, longitudinally, and in shear, respectively. In the overall the predictions were such that: (a) greatest modulus along longer axis; (b) the greatest mineral/collagen stress ratio along the longer axis of collagen fibers (i.e., greatest relief of stresses acting on collagen); and (c) minimal lateral contraction when fibers are loaded along the longer axis. Overall, the pattern of mineralization as put forth in this model predicts a superior mechanical function along the longer axis of collagen fibers, the direction which is more likely to experience greater stresses.     

Fabrication of gelatin/calcium phosphate composite nanofibrous membranes by biomimetic mineralization

Based on the principles of biomimetic mineralization, biocomposite nanofibrous membranes were fabricated by the growth of CaP crystals on electrospun gelatin nanofibers to mimic both the physical architecture and chemical composition of natural bone ECM. Plenty more CaP crystals formed on the nanofibrous membrane containing Ca(2+) ion precursors, in which these crystals were also observed on the inner side of membrane. The release rate of Ca(2+) ion precursors from the nanofibrous membrane was slower than that of PO(4)(3-) ion precursors, suggesting the existence of more strong intermolecular interaction between gelatin and Ca(2+) ions. ATR-FTIR and XRD results clearly revealed the formation of CaP crystals mixed with apatite and CaCO(3), or apatite and TCP on the membranes. The Ca/P molar ratio of crystals obtained from the XPS data was 2.03 and 1.60, which depended on the mineralization conditions. Higher amount of CaP crystals significantly accelerated the deposit rate of bone-like apatite on the surface of composite membrane, meaning to the improved in vivo bone bioactivity.     

Bone material properties and mineral matrix contributions to fracture risk or age in women and men

The strength of bone is related to its mass and geometry, but also to the physical properties of the tissue itself. Bone tissue is composed primarily of collagen and mineral, each of which changes with age, and each of which can be affected by pharmaceutical treatments designed to prevent or reverse the loss of bone. With age, there is a decrease in collagen content, which is associated with an increased mean tissue mineralization, but there is no difference in cross-link levels compared to younger adult bone. In osteoporosis, however, there is a decrease in the reducible collagen cross-links without an alteration in collagen concentration; this would tend to increase bone fragility. In older people, the mean tissue age (MTA) increases, causing the tissue to become more highly mineralized. The increased bone turnover following menopause may reduce global MTA, and would reduce overall tissue mineralization. Bone strength and toughness are positively correlated to bone mineral content, but when bone tissue becomes too highly mineralized, it tends to become brittle. This reduces its toughness, and makes it more prone to fracture from repeated loads and accumulated microcracking. Most approved pharmaceutical treatments for osteoporosis suppress bone turnover, increasing MTA and mineralization of the tissue. This might have either or both of two effects. It could increase bone volume from refilling of the remodeling space, reducing the risk for fracture. Alternatively, the increased MTA could increase the propensity to develop microcracks, and reduce the toughness of bone, making it more likely to fracture. There may also be changes in the morphology of the mineral crystals that could affect the homogeneity of the tissue and impact mechanical properties. These changes might have large positive or negative effects on fracture incidence, and could contribute to the paradox that both large and small increases in density have about the same effect on fracture risk. Bone mineral density measured by DXA does not discriminate between density differences caused by volume changes, and those caused by changes in mineralization. As such, it does not entirely reflect material property changes in aging or osteoporotic bone that contribute to bone's risk for fracture.     

Vitamin K2, a gamma-carboxylating factor of gla-proteins, normalizes the bone crystal nucleation impaired by Mg-insufficiency

It has been reported that the Mg-insufficient bone is fragile upon mechanical loading, despite its high bone mineral density, while vitamin K2 (MK-4: menatetrenone) improved the mechanical strength of Mg-insufficient bone. Therefore, we aimed to elucidate the ultrastructural properties of bone in rats with dietary Mg insufficiency with and without MK-4 supplementation. Morphological examinations including histochemistry, transmission electron microscopy, electron probe microanalysis (EPMA) and X-ray diffraction were conducted on the femora and tibiae of 4-week-old Wistar male rats fed with 1) a normal diet (control group, 0.09% Mg), 2) a Mg-insufficient diet (low Mg group, 0.006% Mg), or 3) a Mg-insufficient diet supplemented with MK-4 (MK-4 group, 0.006% Mg, 0.03% MK-4). MK-4 appeared to inhibit the osteoclastic bone resorption that is stimulated by Mg insufficiency. EPMA analysis, however, revealed an increased concentration of Ca paralleling Mg reduction in the low Mg group. Assessment by X-ray diffraction revealed an abundance of a particular synthetic form of hydroxyapatite in the low Mg group, while control bones featured a variety of mineralized crystals. In addition, Mg-deficient bones featured larger mineral crystals, i.e., crystal overgrowth. This crystalline aberration in Mg-insufficient bones induced collagen fibrils to mineralize easily, even in the absence of mineralized nodules, which therefore led to an early collapse of the fibrils. MK-4 prevented premature collagen mineralization by normalizing the association of collagen fibrils with mineralized nodules. Thus, MK-4 appears to rescue the impaired collagen mineralization caused by Mg insufficiency by promoting a re-association of the process of collagen mineralization with mineralized nodules.     

Haemopoietic long-term bone marrow cultures from adult mice show osteogenic capacity in vitro on 3–dimensional collagen sponges

Abstract. Adult murine bone marrow cells, cultured under conditions for long-term haemopoietic marrow cultures, produce bone matrix proteins and mineralized tissue in vitro , but only after the adherent stromal cells were loaded on a 3-dimensional collagen sponge. Provided more than 8 × 10⁶ cells are loaded, mineralization as measured by ⁸⁵Sr uptake from the culture medium, occurred in this 3-dimensional configuration (3-D) within 6 days. In contrast if undisrupted marrow fragments (containing more than 10⁷ cells) are placed directly on a collagen sponge, then it requires more than 10 days before significant mineralization can similarly be detected. The 2-dimensional (2-D) long-term marrow culture system allows prior expansion of the stromal cells and some differentiation in an osteogenic direction within the adherent stromal layer. This is suggested by the presence of type I collagen and alkaline phos-phatase positive cells. However, synthesis of osteonectin and a bone specific protein, osteocalcin, as well as calcification are only observed in 3-D cultures. Electron microscopy demonstrated hydroxyapatite mineral on collagen fibres, osteoblast-like cells, fibroblasts, cells which accumulated lipids, and macrophages which were retained on the collagen matrices. Irradiation of confluent long-term bone marrow cultures, prior to their loading on the collagen sponge showed that haemopoietic stem cells are not necessary for the mineralization.     

Accelerated Growth Plate Mineralization and Foreshortened Proximal Limb Bones in Fetuin-A Knockout Mice

The plasma protein fetuin-A/alpha2-HS-glycoprotein (genetic symbol Ahsg) is a systemic inhibitor of extraskeletal mineralization, which is best underscored by the excessive mineral deposition found in various tissues of fetuin-A deficient mice on the calcification-prone genetic background DBA/2. Fetuin-A is known to accumulate in the bone matrix thus an effect of fetuin-A on skeletal mineralization is expected. We examined the bones of fetuin-A deficient mice maintained on a C57BL/6 genetic background to avoid bone disease secondary to renal calcification. Here, we show that fetuin-A deficient mice display normal trabecular bone mass in the spine, but increased cortical thickness in the femur. Bone material properties, as well as mineral and collagen characteristics of cortical bone were unaffected by the absence of fetuin-A. In contrast, the long bones especially proximal limb bones were severely stunted in fetuin-A deficient mice compared to wildtype littermates, resulting in increased biomechanical stability of fetuin-A deficient femora in three-point-bending tests. Elevated backscattered electron signal intensities reflected an increased mineral content in the growth plates of fetuin-A deficient long bones, corroborating its physiological role as an inhibitor of excessive mineralization in the growth plate cartilage matrix - a site of vigorous physiological mineralization. We show that in the case of fetuin-A deficiency, active mineralization inhibition is a necessity for proper long bone growth.     

Enhanced cell attachment and osteoblastic activity by P-15 peptide-coated matrix in hydrogels

The cells in bone grow on a composite matrix made up of mineral and organic (mainly type-I collagen) components. In this study, anorganic bone mineral (ABM) particles were coated with a cell-binding domain of type-I collagen (P-15 peptide) to mimic the bone matrix components and suspended in injectable hyaluronate (Hy) hydrogels. The ABM/P-15/Hy was compared to ABM/Hy-the same matrix without P-15 peptide. Osteoblast-like HOS cells migrated through the hydrogels around ABM/P-15 or ABM particles; however, more cells adhered to ABM/P-15/Hy particles, and the cells formed better surface coverage and had more stress fibers on ABM/P-15/Hy. HOS cells cultured on ABM/P-15/Hy had increased osteogenic gene expression for alkaline phosphatase and bone morphogenetic proteins, and deposited more mineralized matrix. Studies with two different hydrogels (carboxymethylcellulose and sodium alginate) showed similar enhanced cell attachment and mineralization. The studies suggest that the ABM/P-15 in hydrogels can be used as an injectable biomimetic matrix to facilitate bone repair.     

Elastic properties of woven bone: effect of mineral content and collagen fibrils orientation

Woven bone is a type of tissue that forms mainly during fracture healing or fetal bone development. Its microstructure can be modeled as a composite with a matrix of mineral (hydroxyapatite) and inclusions of collagen fibrils with a more or less random orientation. In the present study, its elastic properties were estimated as a function of composition (degree of mineralization) and fibril orientation. A self-consistent homogenization scheme considering randomness of inclusions’ orientation was used for this purpose. Lacuno-canalicular porosity in the form of periodically distributed void inclusions was also considered. Assuming collagen fibrils to be uniformly oriented in all directions led to an isotropic tissue with a Young’s modulus \(E = 1.90\) GPa, which is of the same order of magnitude as that of woven bone in fracture calluses. By contrast, assuming fibrils to have a preferential orientation resulted in a Young’s modulus in the preferential direction of 9–16 GPa depending on the mineral content of the tissue. These results are consistent with experimental evidence for woven bone in foetuses, where collagen fibrils are aligned to a certain extent.     

Cadmium Is More Toxic on Volume Bone Mineral Density than Tissue Bone Mineral Density

It has been showed that Cd induces low areal bone mineral density, but we do not know the effect of Cd on cubic bone density. This study was aimed to investigate the effects of Cd on volumetric bone mineral density (VBMD) and tissue bone mineral density (TBMD) in male rats. Twenty-four Sprague-Dawley male rats were randomly divided into four groups that were given cadmium chloride by subcutaneous injection at doses of 0, 0.1, 0.5, and 1.5?mg/kg body weight for 8?weeks, respectively. Then, microcomputed tomography scanning was performed on the proximal tibia, and region of interest was reconstructed using microview software. The VBMD, bone volume fraction of rats treated with 1.5?mg Cd/kg, were significantly decreased compared to control (p?相似文献   

Biomimetically mineralized salmon collagen scaffolds for application in bone tissue engineering

Biomimetic mineralization of collagen is an advantageous method to obtain resorbable collagen/hydroxy-apatite composites for application in bone regeneration. In this report, established procedures for mineralization of bovine collagen were adapted to a new promising source of collagen from salmon skin challenged by the low denaturation temperature. Therefore, in the first instance, variation of temperature, collagen concentration, and ionic strength was performed to reveal optimized parameters for fibrillation and simultaneous mineralization of salmon collagen. Porous scaffolds from mineralized salmon collagen were prepared by controlled freeze-drying and chemical cross-linking. FT-IR analysis demonstrated the mineral phase formed during the preparation process to be hydroxyapatite. The scaffolds exhibited interconnecting porosity, were sufficiently stable under cyclic compression, and showed elastic mechanical properties. Human mesenchymal stem cells were able to adhere to the scaffolds, cell number increased during cultivation, and osteogenic differentiation was demonstrated in terms of alkaline phosphatase activity.