Effects of pH on the microarchitecture of carbonate apatite granules fabricated through a dissolution-precipitation reaction.

Both the composition and architecture of artificial bone govern bone regeneration. Herein, carbonate apatite (CAp), which has a similar mineral composition to bone, was prepared by immersing calcium carbonate (CaCO3) in a phosphate solution with varying acidification levels (pH 6.0) to pH 8.9, to reveal the influence of pH on the composition and architecture of the resultant CAp granules. The composition, crystal morphology, and architecture of resultant CAp granules was well-characterized by X-ray diffraction, scanning electron microscopy, mercury intrusion porosimetry and so on. Consequently, the rate of compositional transformation from CaCO3 to CAp was much higher at pH 6.0 and pH 7.0 than pH 8.0 and pH 8.9. The pH of the phosphate solution did not affect the macroarchitecture of the resultant CAp granules. In contrast, the composition, crystal morphology, microarchitecture, and degradation behavior of the resultant CAp granules were affected by pH of the phosphate solution. In particular, the open-pore distributions and volumes of the CAp granules prepared at pH 6.0-8.9 were changed to reflect the microarchitecture of the samples. Therefore, this study revealed that the pH-controlled elution precipitation reaction is useful for controlling the composition, crystal morphology, microarchitecture, and degradation behavior of the resultant CAp, while preserving its macroarchitecture. Our findings provide fundamental insights into the design of artificial bones for bone regeneration.

In vivo trial of bioresorbable mesh cages contained bone graft granules in rabbit femoral bone defects.

Bone graft granules implanted in bone defects come into physical contact with the host bone and form interconnected porous structure. However, there exists an accidental displacement of granules to unintended locations and leakage of granules from bone defects. Although covering the defect with a barrier membrane prevents granule emanation, this procedure is troublesome. To resolve these problems, we fabricated bioresorbable mesh cages (BRMc) in this study. Bone graft granules composed of carbonate apatite alone (Gr) and bioresorbable mesh cages (BRMc/Gr) introduced the bone graft granules and were implanted into the bone defect in the rabbit femur. Micro-computed tomography and histological analysis were conducted at 4 and 12 weeks after implantation. Osteoprogenitors in the bloodstream from the host bone passed through the pores of BRMc, penetrated the porous structure of graft granules, and might interact with individual granules. Then bone remodeling could progress actively and new bone was formed. The new bone formation was similar to the host bone at 12 weeks and there were minimal signs of local tissue inflammation. BRMc/Gr could reduce the risk of unwanted new bone formation occurring due to loss of granules from the bone defects compared with Gr because BRMc enclosed granules and prevent granules leakage from bone defects and BRMc could not induce unfavorable effects to forme new bone. Additionally, BRMc/Gr could keep granules assembled in one place, avoid displacement of granules to unintended locations, and carry easily. These results demonstrated that BRMc/Gr was effective in bone regeneration and improved clinical handling.

Antibacterial and osteogenic thin films on Ti-6Al-4V surface formed by passivation process in copper hydroxide solution.

Implant-associated infections are threatening and devastating complications that lead to bone destruction and loss. As a smooth surface is suitable for inhibiting bacterial adhesion, endowing antibacterial activity to the Ti surface without any structural changes in the surface topography is an effective strategy for preventing infection. The thin film on the Ti-6Al-4 V surface was functionalized to endow antibacterial activity by immersion in a Cu(OH)2 solution. The resulting surface maintains the surface topography with a surface roughness of 0.03 µm even after the immersion in the Cu(OH)2 solution. Moreover, Cu was detected at approximately 10 atom% from the surface and was present up to a depth of 30 nm of thin film. In vitro experiments revealed that the resulting surface exhibited antibacterial activity against methicillin-resistant Staphylococcus aureus and allowed the cellular proliferation, differentiation, and calcification of MC3T3-E1 cells. Furthermore, in vivo experiments determined that the presence of Cu in the thin film on the Ti-6Al-4 V surface led to no inflammatory reactions, including bone resorption. Thus, immersion in a Cu(OH)2 solution incorporates and immobilizes Cu into the thin film on the Ti-6Al-4 V surface without any structural alternations in the surface topography, and the resulting smooth surface exhibits antibacterial activity and osteogenic cell compatibility without cytotoxicity or inflammatory reactions. Our findings provide fundamental insights into the surface design of Ti-based medical devices, to achieve bone reconstruction and infection prevention.

Passivation of Ti-6Al-4V in Cu(OH)2 solution endowed smooth thin film with antibacterial activity and osteogenic cell compatibility for potentially achieving both bone reconstruction and infection prevention.

Transformable Carbonate Apatite Chains as a Novel Type of Bone Graft.

The aging global population is generating an ever-increasing demand for bone regeneration. Various materials, including blocks, granules, and sponges, are developed for bone regeneration. However, blocks require troublesome shaping and exhibit poor bone-defect conformities; granules migrate into the surrounding tissues during and after filling of the defect, causing handling difficulties and complications; and sponges contain polymers that are subject to religious restrictions, lack osteoconductivity, and may cause inflammation and allergies. Herein, carbonate apatite chains that overcome the limitations of conventional materials are presented. Although carbonate apatite granules migrate, causing inflammation and ectopic calcification, the chains remain in the defects without causing any complications. The chains conform to the defect shape and transform into 3D porous structures, resulting in faster bone regeneration than that observed using granules. Thus, these findings indicate that even traditional calcium phosphates materials can be converted to state-of-the-art materials via shape control.

Effects of Space Dimensionality within Scaffold for Bone Regeneration with Large and Oriented Blood Vessels.

The internal structure of the scaffolds is a key factor for bone regeneration. In this study, we focused on the space dimensionality within the scaffold that may control cell migration and evaluated the effects on the size and orientation of blood vessels and the amount of bone formation in the scaffold. The carbonate apatite scaffolds with intrascaffold space allowing one-dimensional (1D), two-dimensional (2D), or three-dimensional (3D) cell migration were fabricated by 3D printing. These scaffolds had the same space size, i.e., distances between the struts (~300 µm). The scaffolds were implanted into the medial condyle of rabbit femurs for four weeks. Both the size and orientation degree of the blood vessels formed in the scaffolds allowing 1D cell migration were 2.5- to 4.0-fold greater than those of the blood vessels formed in the scaffolds allowing 2D and 3D cell migration. Furthermore, the amount of bone formed in the scaffolds allowing 1D cell migration was 1.4-fold larger than that formed in the scaffolds allowing 2D and 3D cell migration. These are probably because the 1D space limited the direction of cell migration and prevented the branching of blood vessels, whereas 2D and 3D spaces provided the opportunity for random cell migration and blood vessel branching. Thus, scaffolds with 1D space are advantageous for inducing large and oriented blood vessels, resulting in a larger amount of bone formation.

Electrochemical Deposition of Copper on Bioactive Porous Titanium Dioxide Layer: Antibacterial and Pro-Osteogenic Activities.

Ti surfaces must exhibit antibacterial activity without cytotoxicity to promote bone reconstruction and prevent infection simultaneously. In this study, we employed a two-step electrochemical treatment process, namely, microarc oxidation (MAO) and cathodic electrochemical deposition (CED), to modify Ti surfaces. During the MAO step, a porous TiO2 (pTiO2) layer with a surface roughness of approximately 2.0 µm was generated on the Ti surface, and in the CED step, Cu was deposited onto the pTiO2 layer on the Ti surface, forming Cu@pTiO2. Cu@pTiO2 exhibited a similar structure, adhesion strength, and crystal phase to pTiO2. Moreover, X-ray photoelectron spectroscopy (XPS) confirmed the presence of Cu in Cu@pTiO2 at an approximate concentration of 1.0 atom %. Cu@pTiO2 demonstrated a sustained release of Cu ions for a minimum of 28 days in a simulated in vivo environment. In vitro experiments revealed that Cu@pTiO2 effectively eradicated approximately 99% of Staphylococcus aureus and Escherichia coli and inhibited biofilm formation, in contrast to the Ti and pTiO2 surfaces. Moreover, Cu@pTiO2 supported the proliferation of osteoblast-like cells at a rate comparable to that observed on the Ti and pTiO2 surfaces. Similar to pTiO2, Cu@pTiO2 promoted the calcification of osteoblast-like cells compared with Ti. In summary, we successfully conferred antibacterial and pro-osteogenic activities to Ti surfaces without inducing cytotoxic effects or structural and mechanical alterations in pTiO2 through the application of MAO and CED processes. Moreover, we found that the pTiO2 layer promoted bacterial growth and biofilm formation more effectively than the Ti surface, highlighting the potential drawbacks of rough and porous surfaces. Our findings provide fundamental insights into the surface design of Ti-based medical devices for bone reconstruction and infection prevention.

Superiority of Triply Periodic Minimal Surface Gyroid Structure to Strut-Based Grid Structure in Both Strength and Bone Regeneration.

The aging population has rapidly driven the demand for bone regeneration. The pore structure of a scaffold is a critical factor that affects its mechanical strength and bone regeneration. Triply periodic minimal surface gyroid structures similar to the trabecular bone structure are considered superior to strut-based lattice structures (e.g., grids) in terms of bone regeneration. However, at this stage, this is only a hypothesis and is not supported by evidence. In this study, we experimentally validated this hypothesis by comparing gyroid and grid scaffolds composed of carbonate apatite. The gyroid scaffolds possessed compressive strength approximately 1.6-fold higher than that of the grid scaffolds because the gyroid structure prevented stress concentration, whereas the grid structure could not. The porosity of gyroid scaffolds was higher than that of the grid scaffolds; however, porosity and compressive strength generally have a trade-off relationship. Moreover, the gyroid scaffolds formed more than twice the amount of bone as grid scaffolds in a critical-sized bone defect in rabbit femur condyles. This favorable bone regeneration using gyroid scaffolds was attributed to the high permeability (i.e., larger volume of macropores or porosity) and curvature profile of the gyroid structure. Thus, this study validated the conventional hypothesis using in vivo experiments and revealed factors that led to this hypothetical outcome. The findings of this study are expected to contribute to the development of scaffolds that can achieve early bone regeneration without sacrificing the mechanical strength.

Gear-shaped carbonate apatite granules with a hexagonal macropore for rapid bone regeneration.

Synthetic bone grafts are in high demand owing to increased age-related bone disorders in the global aging population. Here, we report fabrication of gear-shaped granules (G-GRNs) for rapid bone healing. G-GRNs possessed six protrusions and a hexagonal macropore in the granular center. These were composed of carbonate apatite, i.e., bone mineral, microspheres with ∼1-µm micropores in the spaces between the microspheres. G-GRNs formed new bone and blood vessels (both on the granular surface and within the macropores) 4 weeks after implantation in the rabbit femur defects. The formed bone structure was similar to that of cancellous bone. The bone percentage in the defect recovered to that in a normal rabbit femur at week-4 post-implantation, and the bone percentage remained constant for the following 8 weeks. Throughout the entire period, the bone percentage in the G-GRN-implanted group was ∼10% higher than that of the group implanted with conventional carbonate apatite granules. Furthermore, a portion of the G-GRNs resorbed at week-4, and resorption continued for the following 8 weeks. Thus, G-GRNs are involved in bone remodeling and are gradually replaced with new bone while maintaining a suitable bone level. These findings provide a basis for the design and fabrication of synthetic bone grafts for achieving rapid bone regeneration.

Fabrication and histological evaluation of ant-nest type porous carbonate apatite artificial bone using polyurethane foam as a porogen.

The composition of carbonate apatite (CO3 Ap) aids bone regeneration. Other features, such as porosity and pore interconnectivity of artificial bone, also govern bone regeneration. In general, a trade-off exists between the porosity and mechanical strength of artificial bone. Therefore, this suggests that the interconnected pores in the ant-nest-type porous (ANP) structure of artificial bone accelerate bone regeneration by minimizing the sacrifice of mechanical strength. The unique structure of polyurethane foam has the potential to endow CO3 Ap with an ANP structure without forming excess pores. This study investigated the efficacy of polyurethane foam as a porogen in providing ANP structure to CO3 Ap artificial bone. The polyurethane foam was completely decomposed by sintering and the resulting CO3 Ap displayed ANP structure with a compressive strength of approximately 15 MPa. Furthermore, in vivo experiments revealed that the migration of cells and tissues into the interior of CO3 Ap through the interconnected pores accelerated bone regeneration in the ANP-structured CO3 Ap. Thus, this indicates that using polyurethane foam as a porogen endows the CO3 Ap artificial bone with an ANP structure that accelerates bone regeneration.

Channel Aperture Characteristics of Carbonate Apatite Honeycomb Scaffolds Affect Ingrowths of Bone and Fibrous Tissues in Vertical Bone Augmentation.

Synthetic scaffolds with the ability to prevent fibrous tissue penetration and promote bone augmentation may realize guided bone regeneration without the use of a barrier membrane for dental implantation. Here, we fabricated two types of honeycomb scaffolds of carbonate apatite, a bone mineral analog, whose channel apertures were square (HC-S) and rectangular (HC-R). The side lengths of the HC-Ss and HC-Rs were 265.8 ± 8.9; 817.7 ± 2.4 and 267.1 ± 5.2 µm, respectively. We placed cylindrical HC-Ss and HC-Rs on the rabbit calvaria. At 4 weeks post-implantation, the HC-Ss prevented fibrous tissue penetration from the top face via the channels, which allowed the new bone to reach the top of the scaffold from the bottom face or the calvarium. In contrast, in the HC-Rs, fibrous tissues filled the channels in the top region. At 12 weeks post-implantation, the HC-Ss were partially replaced with new bone. In the top region of the HC-Rs, although new bone had formed, fibrous tissue remained. According to the findings here and in our previous study, the longer side length rather than the shorter side length of a rectangular scaffold channel aperture is the dominant factor that affects fibrous tissue penetration and new bone augmentation. Furthermore, even though channel aperture areas are similar, bone and fibrous tissue ingrowths are different when the aperture shapes are different.

Structurally optimized honeycomb scaffolds with outstanding ability for vertical bone augmentation.

INTRODUCTION: Cases of intractable dental implant require vertical bone augmentation; however, the predicted bone height and volume are difficult to obtain. In vertical bone augmentation, the contact surface between the scaffold and the bone is limited to the bottom face of the scaffold. Furthermore, the strength decrease caused by scaffold resorption leads to collapse of the augmented site, leading to a decrease in the bone volume and height. OBJECTIVES: To promote bone ingrowth, we fabricated carbonate apatite (i.e., bone mineral) honeycomb (HC) scaffolds with uniaxial channels vertically penetrating the scaffold. Furthermore, we controlled the scaffold resorption rate, eventually the endurability for compression, and the bone height and volume by controlling the strut thickness. METHODS: The channel aperture was controlled to be 230-260 µm to promote bone ingrowth. Furthermore, the strut thicknesses of the HC scaffolds were adjusted to 100, 200, and 300 µm to control the scaffold resorption; these scaffolds were designated as HC100, HC200, and HC300, respectively. RESULTS: At 4 weeks post-implantation on rabbit calvarium, all scaffolds had already vertically augmented new bone close to the top surface of the scaffold. In the following 8 weeks, the height and amount of new bone in all scaffolds increased. Notably, HC300 was resorbed synchronously with new bone formation, allowing it to endure the compression from the fasciae for 12 weeks post-implantation. Furthermore, HC300 formed larger-diameter blood vessels than those of HC100 and HC200. CONCLUSION: The HC scaffolds surpassed the various combined scaffolds and growth factors or stem cells in the ability for vertical bone augmentation. Thus, the HC structure is inherently suitable for vertical bone augmentation. Notably, the HC scaffolds with 300-µm-thick struts enhanced both new bone formation and angiogenesis. This study revealed a structurally suitable design for achieving an outstanding outcome in vertical bone augmentation.

Surface functionalization with copper endows carbonate apatite honeycomb scaffold with antibacterial, proangiogenic, and pro-osteogenic activities.

Osteomyelitis is a potentially devastating inflammatory bone disease that leads to bone destruction and loss. Treatment of osteomyelitis requires the removal of residual bacteria as well as osteogenesis with angiogenesis at the site of treatment. Use of an appropriate amount of copper (Cu) in treatment scaffolds may achieve these goals without the risk of toxicity. In this study, the surface of the carbonate apatite honeycomb scaffold was functionalized with Cu through a dissolution-precipitation reaction. The resulting scaffolds retained the honeycomb structure after immersion in CuCl2 solution, and Cu was precipitated on the surface as libethenite [Cu2(OH)PO4]. The surface Cu concentration was controlled by the concentration of the CuCl2 solution. Scaffolds with a surface Cu concentration of 23.8 wt% exhibited antibacterial and cytotoxic effects, whereas those with concentrations of ≤4.6 wt% exerted antibacterial effects without negatively affecting the cellular adhesion, proliferation, differentiation, and calcification of osteoblast-like cells. Furthermore, scaffolds with a surface Cu concentration of 4.6 wt% Cu inhibited bacterial growth for at least 28 days and displayed proangiogenic and pro-osteogenic activities in vivo. These data confirm the success in functionalizing scaffolds with Cu that may be utilized as an innovative osteomyelitis therapy.

Effects of Scaffold Shape on Bone Regeneration: Tiny Shape Differences Affect the Entire System.

Although studies on scaffolds for tissue generation have mainly focused on the chemical composition and pore structure, the effects of scaffold shape have been overlooked. Scaffold shape determines the scaffold surface area (SA) at the single-scaffold level (i.e., microscopic effects), although it also affects the amount of interscaffold space in the tissue defect at the whole-system level (i.e., macroscopic effects). To clarify these microscopic and macroscopic effects, this study reports the osteogenesis abilities of three types of carbonate apatite granular scaffolds with different shapes, namely, irregularly shaped dense granules (DGs) and two types of honeycomb granules (HCGs) with seven hexagonal channels (∼255 µm in length between opposite sides). The HCGs possessed either 12 protuberances (∼75 µm in length) or no protuberances. Protuberances increased the SA of each granule by 3.24 mm2 while also widening interscaffold spaces and increasing the space percentage in the defect by ∼7.6%. Interscaffold spaces were lower in DGs than HCGs. On DGs, new bone formed only on the surface, whereas on HCGs, bone simultaneously formed on the surface and in intrascaffold channels. Interestingly, HCGs without protuberances formed approximately 30% more new bone than those with protuberances. Thus, even tiny protuberances on the scaffold surface can affect the percentage of interscaffold space, thereby exerting dominant effects on osteogenesis. Our findings demonstrate that bone regeneration can be improved by considering macroscopic shape effects beyond the microscopic effects of the scaffold.

Effects of pore interconnectivity on bone regeneration in carbonate apatite blocks.

Porous architecture in bone substitutes, notably the interconnectivity of pores, is a critical factor for bone ingrowth. However, controlling the pore interconnectivity while maintaining the microarchitecture has not yet been achieved using conventional methods, such as sintering. Herein, we fabricated a porous block using the crystal growth of calcium sulfate dihydrate, and controlled the pore interconnectivity by limiting the region of crystal growth. The calcium sulfate dihydrate blocks were transformed to bone apatite, carbonate apatite (CO3Ap) through dissolution-precipitation reactions. Thus, CO3Ap blocks with 15% and 30% interconnected pore volumes were obtained while maintaining the microarchitecture: they were designated as CO3Ap-15 and CO3Ap-30, respectively. At 4 weeks after implantation in a rabbit femur defect, new bone formed throughout CO3Ap-30, whereas little bone was formed in the center region of CO3Ap-15. At 12 weeks after implantation, a large portion of CO3Ap-30 was replaced with new bone and the boundary with the host bone became blurred. In contrast, CO3Ap-15 remained in the defect and the boundary with the host bone was still clear. Thus, the interconnected pores promote bone ingrowth, followed by replacement of the material with new bone. These findings provide a useful guide for designing bone substitutes for rapid bone regeneration.

Granular honeycomb scaffolds composed of carbonate apatite for simultaneous intra- and inter-granular osteogenesis and angiogenesis.

Granular porous calcium phosphate scaffolds are used for bone regeneration in dentistry. However, in conventional granules, the macropore interconnectivity is poor and has varying size. Herein, we developed a productive method for fabricating carbonate apatite honeycomb granules with uniformly sized macropores based on extrusion molding. Each honeycomb granule possesses three hexagonal macropores of ∼290 â€‹µm along its diagonal. Owing to these macropores, honeycomb granules simultaneously formed new and mature bone and blood vessels in both the interior and exterior of the granules at 4 weeks after implantation. The honeycomb granules are useful for achieving rapid osteogenesis and angiogenesis.

Fabrication and histological evaluation of porous carbonate apatite blocks using disodium hydrogen phosphate crystals as a porogen and phosphatization accelerator.

The porous architecture of artificial bones plays a pivotal role in bone ingrowth. Although salt leaching methods produce predictable porous architectures, their application in the low-temperature fabrication of ceramics remains a challenge. Carbonate apatite (CO3 Ap) blocks with three ranges of pore sizes: 100-200, 200-400, and 400-600 µm, were fabricated from CaCO3 blocks with embedded Na2 HPO4 crystals as a porogen and accelerator for CaCO3 -to-CO3 Ap conversion. CaCO3 blocks were obtained from Ca(OH)2 compacts with Na2 HPO4 by CO2 flow at 100% humidity. When carbonated under 100% water humidity, the dissolution of Na2 HPO4 and the formation of hydroxyapatite were observed. Using 90% methanol and 10% water were beneficial in avoiding the Na2 HPO4 consumption and generating the metastable CaCO3 vaterite, which was rapidly converted into CO3 Ap in a Na2 HPO4 solution in 7 days. For the histological evaluation, the CO3 Ap blocks were implanted in rabbit femur defects. Four weeks after implantation, new bone was formed at the edges of the blocks. After 12 weeks, new bone was observed in the central areas of the material. Notably, CO3 Ap blocks with pore sizes of 100-200 µm were the most effective, exhibiting approximately 23% new bone area. This study sheds new light on the fabrication of tailored porous blocks and provides a useful guide for designing artificial bones.

No-Observed-Effect Level of Silver Phosphate in Carbonate Apatite Artificial Bone on Initial Bone Regeneration.

Fracture-related infections require both treatments for bacteria removal and bone reconstruction. The use of combined broad-spectrum antibacterial silver compounds and artificial bone with high osteogenic activity is considered to be an effective strategy for achieving these treatments in one surgery. However, silver compounds are toxic for living tissues even at low concentrations. Herein, we investigated the no-observed-effect level (NOEL) of silver phosphate (Ag3PO4) in a bone substitute composed of carbonate apatite (CO3Ap), a bone mineral, using in vitro and in vivo experiments. In vitro experiments demonstrated that the CO3Ap artificial bone containing ≥0.1 wt % Ag3PO4 exerted antibacterial effects against Staphylococcus epidermidis, while those containing ≤0.3 wt % Ag3PO4 did not affect cellular adhesion, proliferation, differentiation, and calcification of osteoblast-like MC3T3-E1 cells. In vivo experiments demonstrated that the CO3Ap artificial bone containing ≤0.3 wt % Ag3PO4 replaced a new bone to the same levels as those without Ag3PO4 4 weeks after implantation into the bone defect of the rabbit femur condyle. However, the CO3Ap artificial bone containing 0.3 wt % Ag3PO4 caused an inflammatory reaction, whereas those containing ≤0.1 wt % Ag3PO4 did not. Thus, both bone regeneration and infection control without any adverse effects were achieved using the CO3Ap artificial bone containing 0.1 wt % Ag3PO4, indicating that the NOEL of Ag3PO4 was 0.1 wt %. Our results provide an effective strategy for the treatments of fracture-related infections.

Density Functional Theory-Based Calculation Shed New Light on the Bizarre Addition of Cysteine Thiol to Dopaquinone.

Two types of melanin pigments, brown to black eumelanin and yellow to reddish brown pheomelanin, are biosynthesized through a branched reaction, which is associated with the key intermediate dopaquinone (DQ). In the presence of l-cysteine, DQ immediately binds to the -SH group, resulting in the formation of cysteinyldopa necessary for the pheomelanin production. l-Cysteine prefers to bond with aromatic carbons adjacent to the carbonyl groups, namely C5 and C2. Surprisingly, this Michael addition takes place at 1,6-position of the C5 (and to some extent at C2) rather than usually expected 1,4-position. Such an anomaly on the reactivity necessitates an atomic-scale understanding of the binding mechanism. Using density functional theory-based calculations, we investigated the binding of l-cysteine thiolate (Cys-S-) to DQ. Interestingly, the C2-S bonded intermediate was less energetically stable than the C6-S bonded case. Furthermore, the most preferred Cys-S--attacked intermediate is at the carbon-carbon bridge between the two carbonyls (C3-C4 bridge site) but not on the C5 site. This structure allows the Cys-S- to migrate onto the adjacent C5 or C2 with small activation energies. Further simulation demonstrated a possible conversion pathway of the C5-S (and C2-S) intermediate into 5-S-cysteinyldopa (and 2-S-cysteinyldopa), which is the experimentally identified major (and minor) product. Based on the results, we propose that the binding of Cys-S- to DQ proceeds via the following path: (i) coordination of Cys-S- to C3-C4 bridge, (ii) migration of Cys-S- to C5 (C2), (iii) proton rearrangement from cysteinyl -NH3+ to O4 (O3), and (iv) proton rearrangement from C5 (C2) to O3 (O4).

Granular Honeycombs Composed of Carbonate Apatite, Hydroxyapatite, and ß-Tricalcium Phosphate as Bone Graft Substitutes: Effects of Composition on Bone Formation and Maturation.

Material composition and porous structure are important factors in the formation and maturation of newly formed bone and replacement of materials by new bone. Conventional bone graft materials often lack suitability for bone generation because of the complexity of their macroporous structures, which can interfere with the penetration of cells related to bone remodeling and angiogenesis in the materials. In the present study, carbonate apatite (CO3Ap), hydroxyapatite (HAp), and ß-tricalcium phosphate (TCP) honeycomb granules (HCGs) with uniformly sized macropores (∼115 µm) were fabricated. These HCG macropores were arranged in a regular fashion and penetrated straight into the granules. They were implanted into a rabbit femur defect for further evaluation. In the CO3Ap HCG implantation group, mature bone formed within CO3Ap HCG macropores by 4 weeks after grafting, and a large portion of CO3Ap HCGs was replaced by new bone at 12 weeks. By contrast, in the ß-TCP HCG implantation group, new bone was not always formed in the regions after ß-TCP HCG disappearance, and immature bone was present within ß-TCP HCG macropores even after 12 weeks. HAp HCGs were not resorbed, and their macropores were filled with immature bone. The area of mature bone in the CO3Ap HCG implantation group was 3.3 and 1.6 times higher at 4 weeks and 2.2 and 1.7 times higher at 12 weeks compared with the HAp and ß-TCP HCG implantation groups, respectively. Furthermore, the degrees of bone maturation for CO3Ap, HAp, and ß-TCP HCGs were 100, 34, and 64% at 4 weeks, and 100, 54, and 69% at 12 weeks, respectively. Thus, the composition of the HCGs affected bone formation and maturation.

Carbonate Apatite Micro-Honeycombed Blocks Generate Bone Marrow-Like Tissues as well as Bone.

Hematopoietic stem cells form blood cells in bone marrow and reside in niches. Artificial environments that conserve these niches may generate bone marrow. Osteogenesis, angiogenesis, and material resorption must be regulated to create these environments. These processes are controlled by material composition and macro- and microporous structures. Here, three blocks with different micropore structures are fabricated. Carbonate apatite has nearly the same composition as natural human bone and their honeycomb structure facilitates cell penetration and survival. In samples with high microporosity, endosteum-like tissues such as sinusoids form in areas of material resorption and high local calcium concentration. These conditions resemble environments conducive to niche maintenance. Bone marrow-like tissues and megakaryocytes are successfully generated in this environment. Micropore structure is the most critical factor in bone marrow formation; however, the influences of material composition and macropore structure must also be considered. The results of this study may help develop treatments for bone marrow-related diseases and elucidate the components and functions of the hematopoietic stem cell niche.