Injectable macroporous naturally-derived apatite bone cement as a potential trabecular bone substitute.

In this study, we have formulated a novel apatite bone cements derived from natural sources (i.e. eggshell and fishbone) with improved qualities that is, porosity, resorbability, biological activity, and so forth. The naturally-derived apatite bone cement (i.e. FBDEAp) was prepared by mixing hydroxyapatite (synthesized from fishbone) and tricalcium phosphate (synthesized from eggshell) as a solid phase with a liquid phase (a dilute acidic blend of cement binding accelerator and biopolymers like gelatin and chitosan) with polysorbate (as liquid porogen) to get a desired bone cement paste. The prepared cement paste sets within the clinically acceptable setting time (≤20 min), easily injectable (>85%) through hands and exhibits physiological pH stability (7.3-7.4). The pure apatite phased bone cement was confirmed by x-ray diffraction and Fourier transform infrared spectroscopy analyses. The FBDEAp bone cement possesses acceptable compressive strength (i.e. 5-7 MPa) within trabecular bone range and is resorbable up to 28% in simulated body fluid solution within 12 weeks of incubation at physiological conditions. The FBDEAp is macroporous in nature (average pore size ~50-400 µm) with interconnected pores verified by SEM and micro-CT analyses. The FBDEAp showed significantly increased MG63 cell viability (>125% after 72 h), cell adhesion, proliferation, and key osteogenic genes expression levels (up to 5-13 folds) compared to the synthetically derived, synthetic and eggshell derived as well as synthetic and fishbone derived bone cements. Thus, we strongly believe that our prepared FBDEAp bone cement can be used as potential trabecular bone substitute in orthopedics.

Enhanced remineralisation ability and antibacterial properties of sol-gel glass ionomer cement modified by fluoride containing strontium-based bioactive glass or strontium-containing fluorapatite.

OBJECTIVES: This study aimed to compare two types of bioactive additives which were strontium-containing fluorinated bioactive glass (SrBGF) or strontium-containing fluorapatite (SrFA) added to sol-gel derived glass ionomer cement (SGIC). The objective was to develop antibacterial and mineralisation properties, using bioactive additives, to minimize the occurrence of caries lesions in caries disease. METHODS: Synthesized SrBGF and SrFA nanoparticles were added to SGIC at 1 wt% concentration to improve antibacterial properties against S. mutans, promote remineralisation, and hASCs and hDPSCs viability. Surface roughness and ion-releasing behavior were also evaluated to clarify the effect on the materials. Antibacterial activity was measured via agar disc diffusion and bacterial adhesion. Remineralisation ability was assessed by applying the material to demineralised teeth and subjecting them to a 14-day pH cycle, followed by microCT and SEM-EDS analysis. RESULTS: The addition of SrFA into SGIC significantly improved its antibacterial property. SGIC modified with either SrBGF or SrFA additives could similarly induce apatite crystal precipitation onto demineralised dentin and increase dentin density, indicating its ability to remineralise dentin. Moreover, this study also showed that SGIC modified with SrBGF or SrFA additives had promising results on the in vitro cytotoxicity of hASC and hDPSC. SIGNIFICANT: SrFA has superior antibacterial property as compared to SrBGF while demonstrating equal remineralisation ability. Furthermore, the modified SGIC showed promising results in reducing the cytotoxicity of hASCs and hDPSCs, indicating its potential for managing caries.

Janus Membrane with Intrafibrillarly Strontium-Apatite-Mineralized Collagen for Guided Bone Regeneration.

Commercial collagen membranes face difficulty in guided bone regeneration (GBR) due to the absence of hierarchical structural design, effective interface management, and diverse bioactivity. Herein, a Janus membrane called SrJM is developed that consists of a porous collagen face to enhance osteogenic function and a dense face to maintain barrier function. Specifically, biomimetic intrafibrillar mineralization of collagen with strontium apatite is realized by liquid precursors of amorphous strontium phosphate. Polycaprolactone methacryloyl is further integrated on one side of the collagen as a dense face, which endows SrJM with mechanical support and a prolonged lifespan. In vitro experiments demonstrate that the dense face of SrJM acts as a strong barrier against fibroblasts, while the porous face significantly promotes cell adhesion and osteogenic differentiation through activation of calcium-sensitive receptor/integrin/Wnt signaling pathways. Meanwhile, SrJM effectively enhances osteogenesis and angiogenesis by recruiting stem cells and modulating osteoimmune response, thus creating an ideal microenvironment for bone regeneration. In vivo studies verify that the bone defect region guided by SrJM is completely repaired by newly formed vascularized bone. Overall, the outstanding performance of SrJM supports its ongoing development as a multifunctional GBR membrane, and this study provides a versatile strategy of fabricating collagen-based biomaterials for hard tissue regeneration.

Sintered fluorapatite scaffolds as an autograft-like engineered bone graft.

Hydroxyapatite (HA)-based materials are widely used as bone substitutes due to their inherent biocompatibility, osteoconductivity, and bio-absorption properties. However, HA scaffolds lack compressive strength when compared to autograft bone. It has been shown that the fluoridated form of HA, fluorapatite (FA), can be sintered to obtain this desired strength as well as slower degradation properties. Also, FA surfaces have been previously shown to promote stem cell differentiation toward an osteogenic lineage. Thus, it was hypothesized that FA, with and without stromal vascular fraction (SVF), would guide bone healing to an equal or better extent than the clinical gold standard. The regenerative potentials of these scaffolds were tested in 32 Lewis rats in a femoral condylar defect model with untreated (negative), isograft (positive), and commercial HA as controls. Animals were survived for 12 weeks post-implantation. A semi-quantitative micro-CT analysis was developed to quantify the percent new bone formation within the defects. Our model showed significantly higher (p < .05) new bone depositions in all apatite groups compared to the autograft group. Overall, the FA group had the most significant new bone deposition, while the differences between HA, FA, and FA + SVF were insignificant (p > .05). Histological observations supported the micro-CT findings and highlighted the presence of healthy bone tissues without interposing capsules or intense immune responses for FA groups. Most importantly, the regenerating bone tissue within the FA + SVF scaffolds resembled the architecture of the surrounding trabecular bone, showing intertrabecular spaces, while the FA group presented a denser cortical bone-like architecture. Also, a lower density of cells was observed near FA granules compared to HA surfaces, suggesting a reduced immune response. This first in vivo rat study supported the tested hypothesis, illustrating the utility of FA as a bone scaffold material.

A natural biogenic fluorapatite as a new biomaterial for orthopedics and dentistry: antibacterial activity of lingula seashell and its use for nanostructured biomimetic coatings.

Calcium phosphates are widely studied in orthopedics and dentistry, to obtain biomimetic and antibacterial implants. However, the multi-substituted composition of mineralized tissues is not fully reproducible from synthetic procedures. Here, for the first time, we investigate the possible use of a natural, fluorapatite-based material, i.e., Lingula anatina seashell, resembling the composition of bone and enamel, as a biomaterial source for orthopedics and dentistry. Indeed, thanks to its unique mineralization process and conditions, L. anatina seashell is among the few natural apatite-based shells, and naturally contains ions having possible antibacterial efficacy, i.e., fluorine and zinc. After characterization, we explore its deposition by ionized jet deposition (IJD), to obtain nanostructured coatings for implantable devices. For the first time, we demonstrate that L. anatina seashells have strong antibacterial properties. Indeed, they significantly inhibit planktonic growth and cell adhesion of both Gram-positive Staphylococcus aureus and Gram-negative Escherichia coli. The two strains show different susceptibility to the mineral and organic parts of the seashells, the first being more susceptible to zinc and fluorine in the mineral part, and the second to the organic (chitin-based) component. Upon deposition by IJD, all films exhibit a nanostructured morphology and sub-micrometric thickness. The multi-doped, complex composition of the target is maintained in the coating, demonstrating the feasibility of deposition of coatings starting from biogenic precursors (seashells). In conclusion, Lingula seashell-based coatings are non-cytotoxic with strong antimicrobial capability, especially against Gram-positive strains, consistently with their higher susceptibility to fluorine and zinc. Importantly, these properties are improved compared to synthetic fluorapatite, showing that the films are promising for antimicrobial applications.

GelMA/TCP nanocomposite scaffold for vital pulp therapy.

Pulp capping is a necessary procedure for preserving the vitality and health of the dental pulp, playing a crucial role in preventing the need for root canal treatment or tooth extraction. Here, we developed an electrospun gelatin methacryloyl (GelMA) fibrous scaffold incorporating beta-tricalcium phosphate (TCP) particles for pulp capping. A comprehensive morphological, physical-chemical, and mechanical characterization of the engineered fibrous scaffolds was performed. In vitro bioactivity, cell compatibility, and odontogenic differentiation potential of the scaffolds in dental pulp stem cells (DPSCs) were also evaluated. A pre-clinical in vivo model was used to determine the therapeutic role of the GelMA/TCP scaffolds in promoting hard tissue formation. Morphological, chemical, and thermal analyses confirmed effective TCP incorporation in the GelMA nanofibers. The GelMA+20%TCP nanofibrous scaffold exhibited bead-free morphology and suitable mechanical and degradation properties. In vitro, GelMA+20%TCP scaffolds supported apatite-like formation, improved cell spreading, and increased deposition of mineralization nodules. Gene expression analysis revealed upregulation of ALPL, RUNX2, COL1A1, and DMP1 in the presence of TCP-laden scaffolds. In vivo, analyses showed mild inflammatory reaction upon scaffolds' contact while supporting mineralized tissue formation. Although the levels of Nestin and DMP1 proteins did not exceed those associated with the clinical reference treatment (i.e., mineral trioxide aggregate), the GelMA+20%TCP scaffold exhibited comparable levels, thus suggesting the emergence of differentiated odontoblast-like cells capable of dentin matrix secretion. Our innovative GelMA/TCP scaffold represents a simplified and efficient alternative to conventional pulp-capping biomaterials. STATEMENT OF SIGNIFICANCE: Vital pulp therapy (VPT) aims to preserve dental pulp vitality and avoid root canal treatment. Biomaterials that bolster mineralized tissue regeneration with ease of use are still lacking. We successfully engineered gelatin methacryloyl (GelMA) electrospun scaffolds incorporated with beta-tricalcium phosphate (TCP) for VPT. Notably, electrospun GelMA-based scaffolds containing 20% (w/v) of TCP exhibited favorable mechanical properties and degradation, cytocompatibility, and mineralization potential indicated by apatite-like structures in vitro and mineralized tissue deposition in vivo, although not surpassing those associated with the standard of care. Collectively, our innovative GelMA/TCP scaffold represents a simplified alternative to conventional pulp capping materials such as MTA and Biodentine™ since it is a ready-to-use biomaterial, requires no setting time, and is therapeutically effective.

Primary human osteoblast and mesenchymal stem cell responses to apatite/tricalcium phosphate bone cement modified with polyacrylic acid and bioactive glass.

In this work, three different modified cements, control apatite/beta-tricalcium phosphate cement (CPC), polymeric CPC (p-CPC), and bioactive glass added polymeric cement (p-CPC/BG) were evaluated regarding their physical properties and the responses of primary human osteoblast cells (HObs) and mesenchymal stem cells (MSCs). Although polyacrylic acid (PAA) increased compressive strength and Young's modulus of the cement, it could cause poor apatite phase formation, a prolonged setting time, and a lower degradation rate. Consequently, bioactive glass (BG) was added to PAA/cement to improve its physical properties, such as compressive strength, Young's modulus, setting time, and degradation. For in vitro testing, HObs viability was assessed under two culture systems with cement-preconditioned medium (indirect) and with cement (direct). HObs viability was examined in direct contact with cements treated by different prewashing conditions. HObs presented a more well spread morphology on cement soaked in medium overnight, as compared to other cements with no treatment and washing in PBS. In addition, the proliferation, differentiation, and total collagen production of both HObs and MSCs adhered to the cement were detected. Cells showed excellent proliferation on PAA/cement and PAA/BG/cement. Furthermore, the higher released Si ion and lower acidosis of PAA/BG/cement-conditioned medium resulted in an increase in osteogenic differentiation (HObs and MSCs) and enhanced collagen production (HObs in osteogenic medium and MSCs in control medium). Therefore, our findings suggest that BG incorporated PAA/apatite/ß-TCP cement could be a promising formula for bone repair applications.

Design of antibacterial apatitic composite cement loaded with Ciprofloxacin: Investigations on the physicochemical Properties, release Kinetics, and antibacterial activity.

This work aims to develop an injectable and antibacterial composite cement for bone substitution and prevention/treatment of bone infections. This cement is composed of calcium phosphate, calcium carbonate, bioactive glass, sodium alginate, and ciprofloxacin. The effect of ciprofloxacin on the microstructure, chemical composition, setting properties, cohesion, injectability, and compressive strength was investigated. The in vitro drug release kinetics and the antibacterial activity of ciprofloxacin-loaded composites against staphylococcus aureus and Escherichia coli pathogens were investigated. XRD and FTIR analysis demonstrated that the formulated cements are composed of a nanocrystalline carbonated apatite analogous to the mineral part of the bone. The evaluation of the composite cement's properties revealed that the incorporation of 3 and 9 wt% of ciprofloxacin affects the microstructural and physicochemical properties of the cement, resulting in a prolonged setting time, and a slight decrease in injectability and compressive strength. The in vitro drug release study revealed sustained release profiles over 18 days. The amounts of ciprofloxacin released per day (0.2 -15.2 mg/L) depend on the cement composition and the amount of ciprofloxacin incorporated. The antibacterial activity of ciprofloxacin-loaded cement composites attested to their effectiveness to inhibit the growth of Staphylococcus aureus and Escherichia coli.

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.

Apatite nanosheets inhibit initial smooth muscle cell proliferation by damaging cell membrane.

Understanding how nanostructured coatings interact with cells is related to how they manipulate cell behaviors and is therefore critical for designing better biomaterials. The apatite nanosheets were deposited on metallic substrates via biomimetic precipitation. Cell viability of apatite nanosheets towards to smooth muscle cells (SMCs) were investigated, and the underlying mechanism was proposed. Apatite nanosheets presented inhibitory activity on SMC growth, and caused rupture of cell membranes. On the basis of measuring changes in intracellular calcium ([Ca2+]i), observing cell contraction and apatite nanosheets - SMC interaction, it was found that calcium ions released from apatite led to rises in [Ca2+]i, which induced vigorous SMC contraction on apatite nanosheets. Consequently, the cell membrane of individual SMCs was cut/penetrated by the sharp edges of apatite nanosheets, resulting in cell inactivation. This damage of cell membranes suggests a novel mechanism to manipulate cell viability, and may offer insights for the better design of calcium-based nanostructured coatings or other biomedical applications.

Osteoclast behaviors on the surface of deproteinized bovine bone mineral and carbonate apatite substitutes in vitro.

The present study investigated the osteoclast differentiation potential and paracrine effects of osteoclasts on osteoblast differentiation when the cells were cultured directly on two bone substitutes (BSs): deproteinized bovine bone mineral (DBBM) and carbonate apatite (CO3 Ap). Human primary osteoclasts cultured on the BSs were assessed by tartrate-resistant acid phosphatase (TRAP) and actin ring staining. Thereafter, the mRNA levels of osteoclastic differentiation markers were quantified by real-time PCR. Osteoblast behaviors in response to conditioned media collected from osteoclast cultures were investigated. Interestingly, mature osteoclasts were occasionally observed on the surface of the CO3 Ap granules, whereas very few and small osteoclasts were observed on DBBM. Similarly, real-time PCR analysis showed higher mRNA levels of osteoclast markers, including cathepsin K and TRAP, in the cells cultured on CO3 Ap than in those cultured on DBBM. Furthermore, compared to DBBM, CO3 Ap promoted osteoblast differentiation in human primary osteoblasts, whereas few paracrine effects of osteoclasts cultured with either BS were observed on the osteoblast differentiation potential. These limited results showed that CO3 Ap provided a favorable surface for osteoclast differentiation, as well as osteoblasts, compared to DBBM in vitro.

Nanoapatites Doped and Co-Doped with Noble Metal Ions as Modern Antibiofilm Materials for Biomedical Applications against Drug-Resistant Clinical Strains of Enterococcus faecalis VRE and Staphylococcus aureus MRSA.

The main aim of our research was to investigate antiadhesive and antibiofilm properties of nanocrystalline apatites doped and co-doped with noble metal ions (Ag+, Au+, and Pd2+) against selected drug-resistant strains of Enterococcus faecalis and Staphylococcus aureus. The materials with the structure of apatite (hydroxyapatite, nHAp; hydroxy-chlor-apatites, OH-Cl-Ap) containing 1 mol% and 2 mol% of dopants and co-dopants were successfully obtained by the wet chemistry method. The majority of them contained an additional phase of metallic nanoparticles, in particular, AuNPs and PdNPs, which was confirmed by the XRPD, FTIR, UV-Vis, and SEM-EDS techniques. Extensive microbiological tests of the nanoapatites were carried out determining their MIC, MBC value, and FICI. The antiadhesive and antibiofilm properties of the tested nanoapatites were determined in detail with the use of fluorescence microscopy and computer image analysis. The results showed that almost all tested nanoapatites strongly inhibit adhesion and biofilm production of the tested bacterial strains. Biomaterials have not shown any significant cytotoxic effect on fibroblasts and even increased their survival when co-incubated with bacterial biofilms. Performed analyses confirmed that the nanoapatites doped and co-doped with noble metal ions are safe and excellent antiadhesive and antibiofilm biomaterials with potential use in the future in medical sectors.

Optimizing the strontium content to achieve an ideal osseointegration through balancing apatite-forming ability and osteogenic activity.

Implant failure caused by unsatisfying osseointegration is still a noteworthy clinical problem. Strontium (Sr) has been confirmed to be a bioactive element that facilitates bone growth. In this study, Sr was surface incorporated in titanium (Ti) implant with different contents. The XRD results demonstrated that Sr existed mainly in the form of SrTiO3. All Sr-contained implants showed sustainable Sr2+ release behavior. Meanwhile, the Sr2+ release rate was proportional to the Sr content. The in vitro immersing test showed that the apatite-forming ability on the implant surface was decreased with the increase of Sr content. Conversely, the cell experiments manifested that implants with high content of Sr were more favorable to cell spreading, proliferation, osteogenic differentiation, and extracellular matrix mineralization. The in vivo implant experiment revealed that Sr-incorporation could improve osseointegration, new bone formation and mineralization, and bone-implant bonding strength. In addition, Ti5Sr, which possessed a combined good osteogenic activity and apatite-forming ability, exhibited the best in vivo overall performance. In summary, we first put forward the competitive effect of osteogenic activity and apatite-forming ability on bone-implant osseointegration, which would provide a new strategy for implant design.

The effect of simple heat treatment on apatite formation on grit-blasted/acid-etched dental Ti implants already in clinical use.

Grit-blasted/acid-etched titanium dental implants have a moderately roughened surface that is suitable for cell adhesion and exhibits faster osseointegration. However, the roughened surface does not always maintain stable fixation over a long period. In this study, a simple heat treatment at 600°C was performed on a commercially available dental Ti implant with grit-blasting/acid-etching, and its effect on mineralization capacity was assessed by examining apatite formation in a simulated body fluid (SBF). The as-purchased implant displayed a moderately roughened surface at the micrometer scale. Its surface was composed of titanium hydride accompanied by a small amount of alumina particles derived from the grit-blasting. Heat treatment transformed the titanium hydride into rutile without evidently changing the surface morphology. The immersion in SBF revealed that apatite formed on the heated implant at 7 days. Furthermore, apatite formed on the Ti rod surface within 1 day when the metal was subjected to acid and heat treatment without blasting. These indicate that apatite formation was conferred on the commercially available dental implant by simple heat treatment, although its induction period was slightly affected by alumina particles remaining on the implant surface. The heat-treated implant should achieve stronger and more stable bone bonding due to its apatite formation.

Effects of the carbonate content in carbonate apatite on bone replacement.

Carbonate apatite (CO3 Ap), an inorganic component of human bone, has been clinically applied as an artificial bone substitute. In this study, the effects of the CO3 content in CO3 Ap on the replacement by new bone were studied by fabricating CO3 Ap granules containing 0.9-8.3 wt% of CO3 . The dissolution rate of CO3 Ap in a weak acidic solution, mimicking the Howship's lacunae, was rapid for the CO3 Ap granules containing a larger amount of CO3 . Histological analyses demonstrated the rapid resorption in CO3 Ap and replacement by natural bone tissue when the CO3 content was increased. Therefore, the CO3 content in CO3 Ap is a key factor that influences the replacement of the bone tissue.

Highly Porous Fluorapatite/ß-1,3-Glucan Composite for Bone Tissue Regeneration: Characterization and In-Vitro Assessment of Biomedical Potential.

A novel fluorapatite/glucan composite ("FAP/glucan") was developed for the treatment of bone defects. Due to the presence of polysaccharide polymer (ß-1,3-glucan), the composite is highly flexible and thus very convenient for surgery. Its physicochemical and microstructural properties were evaluated using scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR), mercury intrusion, mechanical testing and compared with the reference material, which was a hydroxyapatite/glucan composite ("HAP/glucan") with hydroxyapatite granules (HAP) instead of FAP. It was found that FAP/glucan has a higher density and lower porosity than the reference material. The correlation between the Young's modulus and the compressive strength between the materials is different in a dry and wet state. Bioactivity assessment showed a lower ability to form apatite and lower uptake of apatite-forming ions from the simulated body fluid by FAP/glucan material in comparison to the reference material. Moreover, FAP/glucan was determined to be of optimal fluoride release capacity for osteoblasts growth requirements. The results of cell culture experiments showed that fluoride-containing biomaterial was non-toxic, enhanced the synthesis of osteocalcin and stimulated the adhesion of osteogenic cells.

Synthesis and characterization of silver substituted strontium phosphate silicate apatite using solid-state reaction for osteoregenerative applications.

Strontium phosphosilicate is one of the fastest-growing apatite in bone regeneration application due to the presence of strontium and silica components in the parent materials. However, bacterial infections cause setbacks to the bone regeneration process often leading to surgical revisions, and is a big issue that needs to be addressed. Silver on this front has proven to be a great substituent as seen in the case of calcium phosphate-based ceramics that addresses the bactericidal properties of a biomaterial. Apatite strontium phosphosilicate substituted with a stoichiometric amount of silver as a dopant was synthesized using a high-temperature solid-state reaction. The phase formation was characterized by XRD and FT-IR coupled with morphological features visualized using Electron Microscopy. Antibacterial properties were investigated quantitatively using Colony-forming unit method against both Gram-positive as well as Gram-negative bacteria. Cytotoxicity assay was performed against MG-63 Cell lines and it showed excellent biocompatibility at 25ug/ml with optimal doping of 2% silver. Further, apatite seeding and formation were characterized after immersion in simulated body fluid solution which showed apatite phase formation initiated after 4 days of treatment characterized by XRD and FT-IR studies. This apatite formation was also visualized and confirmed using SEM.

Reconstruction of critical-size segmental defects in rat femurs using carbonate apatite honeycomb scaffolds.

Critical-size segmental defects are formidable challenges in orthopedic surgery. Various scaffolds have been developed to facilitate bone reconstruction within such defects. Many previously studied scaffolds achieved effective outcomes with a combination of high cost, high-risk growth factors or stem cells. Herein, we developed honeycomb scaffolds (HCSs) comprising carbonate apatite (CO3 Ap) containing 8% carbonate, identical to human bone composition. The CO3 Ap HCSs were white-columned blocks harboring regularly arranged macropore channels of a size and wall thickness of 156 ± 5 µm and 102 ± 10 µm, respectively. The compressive strengths of the HCSs parallel and perpendicular to the macropore channel direction were 51.0 ± 11.8 and 15.6 ± 2.2 MPa, respectively. The HCSs were grafted into critical-sized segmental defects in rat femurs. The HCSs bore high-load stresses without any observed breakage. Two-weeks post-implantation, calluses formed around the HCSs and immature bone formed in the HCS interior. The calluses and immature bone matured until 8 weeks via endochondral ossification. At 12 weeks post-implantation, large parts of the HCSs were gradually replaced by newly formed bone. The bone reconstruction efficacy of the CO3 Ap HCSs alone was comparable to that of protein and cell scaffolds, while achieving a lower cost and increased safety.

Enhanced BMP-2-Mediated Bone Repair Using an Anisotropic Silk Fibroin Scaffold Coated with Bone-like Apatite.

The repair of large bone defects remains challenging and often requires graft material due to limited availability of autologous bone. In clinical settings, collagen sponges loaded with excessive amounts of bone morphogenetic protein 2 (rhBMP-2) are occasionally used for the treatment of bone non-unions, increasing the risk of adverse events. Therefore, strategies to reduce rhBMP-2 dosage are desirable. Silk scaffolds show great promise due to their favorable biocompatibility and their utility for various biofabrication methods. For this study, we generated silk scaffolds with axially aligned pores, which were subsequently treated with 10× simulated body fluid (SBF) to generate an apatitic calcium phosphate coating. Using a rat femoral critical sized defect model (CSD) we evaluated if the resulting scaffold allows the reduction of BMP-2 dosage to promote efficient bone repair by providing appropriate guidance cues. Highly porous, anisotropic silk scaffolds were produced, demonstrating good cytocompatibility in vitro and treatment with 10× SBF resulted in efficient surface coating. In vivo, the coated silk scaffolds loaded with a low dose of rhBMP-2 demonstrated significantly improved bone regeneration when compared to the unmineralized scaffold. Overall, our findings show that this simple and cost-efficient technique yields scaffolds that enhance rhBMP-2 mediated bone healing.

Self-Assembled Type I Collagen-Apatite Fibers with Varying Mineralization Extent and Luminescent Terbium Promote Osteogenic Differentiation of Mesenchymal Stem Cells.

This work explores in depth the simultaneous self-assembly and mineralization of type I collagen by a base-acid neutralization technique to prepare biomimetic collagen-apatite fibrils with varying mineralization extent and doped with luminescent bactericidal Tb3+ ions. Two variants of the method are tested: base-acid titration, a solution of Ca(OH)2 is added dropwise to a stirred solution containing type I collagen dispersed in H3 PO4 ; and direct mixing, the Ca(OH)2 solution is added by fast dripping onto the acidic solution. Only the direct mixing variant yielded an effective control of calcium phosphate polymorphism. Luminescence spectroscopy reveals the long luminescence lifetime and high relative luminescence intensity of the Tb3+ -doped materials, while two-photon confocal fluorescence microscopy shows the characteristic green fluorescence light when using excitation wavelength of 458 nm, which is not harmful to bone tissue. Cytotoxicity/viability tests reveal that direct mixing samples show higher cell proliferation than titration samples. Additionally, osteogenic differentiation essays show that all mineralized fibrils promote the osteogenic differentiation, but the effect is more pronounced when using samples prepared by direct mixing, and more notably when using the Tb3+ -doped mineralized fibrils. Based on these findings it is concluded that the new nanocomposite is an ideal candidate for bone regenerative therapy.