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Graphene-based three-dimensional (3D) porous scaffolds have been extensively investigated in the photothermal treatment of tumor-induced bone defects due to their photothermal and osteogenic capacity. However, scaffold processing destroys conjugated graphene structure and reduces its photothermal conversion efficiency. In this study, a graphene-based 3D scaffold (GS) with intact conjugated structure was prepared by chemical vapor deposition (CVD). GS was rapidly mineralized biomimetically by a newly developed semi-dry electrochemical deposition method to form a hydroxyapatite (HA) incorporated graphene scaffold (HA-GS). The simulation of the charged particle dynamics provides a better understanding of the mechanism of semi-dry electrodeposition. This scaffold exhibits high photothermal sensitivity that generates sufficient thermal energy for photothermal therapy even under near-infrared irradiation (980 nm) with extremely low power density (0.2 W/cm

Injectable hydrogels based on various functional biocompatible materials have made rapid progress in the field of bone repair. In this study, a self-healing and injectable polysaccharide-based hydrogel was prepared for bone tissue engineering. The hydrogel was made of carboxymethyl chitosan (CMCS) and calcium pre-cross-linked oxidized gellan gum (OGG) cross-linked by the Schiff-base reaction. Meanwhile, magnetic hydroxyapatite/gelatin microspheres (MHGMs) were prepared by the emulsion cross-linking method. The antibacterial drugs, tetracycline hydrochloride (TH) and silver sulfadiazine (AgSD), were embedded into the MHGMs. To improve the mechanical and biological properties of the hydrogels, composite hydrogels were prepared by compounding hydroxyapatite (HAp) and drug-embedded MHGMs. The physical, chemical, mechanical and rheological properties of the composite hydrogels were characterized, as well as in vitro antibacterial tests and biocompatibility assays, respectively. Our results showed that the composite hydrogel with 6% (w/v) HAp and 10 mg/mL MHGMs exhibited good magnetic responsiveness, self-healing and injectability. Compared with the pure hydrogel, the composite hydrogel showed a 38.8% reduction in gelation time (196 to 120 s), a 65.6% decrease in swelling rate (39.4 to 13.6), a 51.9% increase in mass residual after degradation (79.5 to 120.8%), and a 143.7% increase in maximum compressive stress (53.6 to 130.6 KPa). In addition, this composite hydrogel showed good drug retardation properties and antibacterial effects against both S. aureus and E. coli. CCK-8 assay showed that composite hydrogel maintained high cell viability (87%) and rapid cell proliferation after 3 days, indicating that this smart hydrogel is expected to be an alternative scaffold for drug delivery and bone regeneration. STATEMENT OF SIGNIFICANCE: Biopolymer hydrogels have been considered as the promising materials for the treatment of tissue engineering and drug delivery. Injectable hydrogels with and self-healing properties and responsiveness to external stimuli have been extensively investigated as cell scaffolds and bone defects, due to their diversity and prolonged lifetime. Magnetism has also been involved in biomedical applications and played significant roles in targeted drug delivery and anti-cancer therapy. We speculate that development of dual cross-linked hydrogels basing biopolymers with multi-functionalities, such as injectable, self-healing, magnetic and anti-bacterial properties, would greatly broaden the application for bone tissue regeneration and drug delivery.

Hydroxyapatite nanoparticles (HAP NPs) are host materials and can be modified with various substrates and dopants. Among them, rare earth (RE) ions doped HAP NPs have gathered attention due to their unique physicochemical and imaging properties. Compared to other fluorescence probes, RE-doped HAP NPs display advantages in high brightness, high contrast, photostability, nonblinking, and narrow emission bands. Meanwhile, their intrinsic features (composition, morphology, size, crystallinity, and luminescence intensity) can be adjusted by changing the dopant ratio, synthesizing temperature, reaction time, and techniques. And they have been used in various biomedical applications, including imaging probe, drug delivery, bone tissue engineering, and antibacterial studies. This review surveys the luminescent properties, fluorescence enhancement, synthetic methods, and biocompatibility of various RE-doped HAP NPs consolidated from different research works, for their employments in biomedical applications. For this literature review, an electronic search was conducted in the Pubmed, Web of Science, Google Scholar, Scopus and SciFinder databases, using the keywords: hydroxyapatite, rare earth, lanthanide, fluorescence, and imaging. Literature searches of English-language publications from 1979 with updates through April, 2022, and a total of 472 potential papers were identified. In addition, a few references were located by noting their citation in other studies reviewed. STATEMENT OF SIGNIFICANCE: Hydroxyapatite nanoparticles (HAP NPs) have a broad range of promising biological applications. Although prospective biomedical applications are not limited to rare earth-doped hydroxyapatite nanoparticles (RE-doped HAP NPs), some cases do make use of the distinctive features of RE-elements to achieve the expected functions for HAP families. This review surveys the luminescent properties, synthetic methods, and biocompatibility of various RE-doped HAP NPs consolidated from different research works, for their employments in biomedical applications, including imaging probe, drug delivery, bone tissue repair and tracking, and anti-bacteria. Overall, we expect to shed some light on broadening the research and application of RE-doped HAP NPs in biomedical field.

Concerns about the potential systematic toxicity limit the extensive application of traditional therapeutic drugs for melanoma therapy, nano-hydroxyapatite (nHA) with good biocompatibility and anti-tumor ability could be an alternative choice. In this study, nHA was employed as an anti-tumor biomaterial due to its tumor-specific toxicity. Meanwhile, granulocyte-macrophage colony-stimulating factor (GM-CSF) served as the immune adjuvant to activate the immune response. The delivery platform was fabricated by co-encapsulation of both nHA and GM-CSF into a biocompatible thermosensitive PLGA-PEG-PLGA hydrogel. The results showed that the bio-activities of nHA and GM-CSF could be well-maintained within the hydrogel. Interestingly, the addition of nHA could attenuate the burst release of GM-CSF due to possible protein absorption capacity of nHA, which is beneficial for GM-CSF sustainable release at the tumor site, achieving boosted and prolonged anti-tumor immunity. The in vitro and in vivo data demonstrated that nHA/GM-CSF hydrogel exhibited greater potency to inhibit tumor growth via enhanced CD8

This study aimed to investigate the accumulation and differentiation of mesenchymal stem cells (MSCs) around octacalcium phosphate (OCP) compared with those around calcium-deficient hydroxyapatite (CDHA), a material obtained through hydrolysis of the original OCP. Leptin receptor (Lepr)-expressing bone marrow-derived MSCs around the OCP and CDHA were pursued utilizing genetically modified Lepr-cre/Tomato mice. OCP and CDHA granules were implanted into the tibia defect of the mice for 10 weeks and subjected to histomorphometric and immunohistochemical analyses. The structural properties of OCP and CDHA after inoculation into mouse subcutaneous tissue (until 4 weeks) or culture mediums (14 days) were analyzed using physicochemical techniques. In vitro osteoblastic differentiation of primary MSCs was examined with the materials for 14 days. While Lepr-cre/Tomato positive cells (red) accumulated around both OCP and CDHA, Lepr and osteocalcin double-positive osteoblastic cells (yellow) were significantly more abundant around OCP than around CDHA in the early implantation period. OCP enhanced the osteoblastic differentiation of MSCs more than CDHA in vitro. Physicochemical and structual analyses provided evidence that OCP tended to convert to the apatitic phase in the tested physiological environments. The higher osteoconductivity of OCP originated from a capacity-enhancing osteoblastic differentiation of committed osteoblast progenitors in bone marrow accompanied by OCP hydrolysis. STATEMENT OF SIGNIFICANCE: MSCs play a key role in bone regeneration through osteoblastic differentiation. Calcium phosphates have been widely applied as bone substitute materials, and OCP has a better ability to promote osteoblast differentiation of MSCs than that of HA in vitro. However, it is not clear how MSCs accumulate in the bone marrow and differentiate into osteoblasts during bone regeneration in vivo. In this study, we focused on the leptin receptor, a marker of bone marrow-derived MSCs. Using genetically modified mice labeled with the red fluorescent protein Tomato, we observed the accumulation of MSCs around calcium phosphates implanted in tibia bone defects and their differentiation into osteoblasts.

Biogenic collagen membranes have been widely used as soft tissue barriers in guided bone regeneration (GBR) and guided tissue regeneration (GTR). Nevertheless, their clinical performance remains unsatisfactory because of their low mechanical strength and fast degradation rate in vivo. Although cross-linking with chemical agents is effective and reliable for prolonging the degradation time of collagen membranes, some adverse effects including potential cytotoxicity and undesirable tissue integration have been observed during this process. As a fundamental nutritional trace element, zinc plays an active role in promoting the growth of cells and regulating the degradation of the collagen matrix. Herein, a biogenic collagen membrane was cross-linked with glutaraldehyde-alendronate to prolong its degradation time. The physiochemical and biological properties were enhanced by the incorporation of zinc-doped nanohydroxyapatite (nZnHA), with the native structure of collagen preserved. Specifically, the cross-linking combined with the incorporation of 1% and 2% nZnHA seemed to endow the membrane with the most appropriate biocompatibility and tissue integration capability among the cross-linked membranes, as well as offering a degradation period of six weeks in a rat subcutaneous model. Thus, improving the clinical performance of biogenic collagen membranes by cross-linking together with the incorporation of nZnHA is a promising strategy for the improvement of biogenic collagen membranes. STATEMENT OF SIGNIFICANCE: The significance of this research includes.

Disadvantages of conventional drug delivery systems (DDS), such as systemic circulation, interaction with physiochemical factors, reduced bioavailability, and insufficient drug concentration at bone defect site, have underlined the importance of developing efficacious local drug delivery systems. Octacalcium phosphate (OCP) is presumed to be the precursor of biologically formed apatite, owing to its similarity to hydroxyapatite (HAp) and readiness to convert to it. Specific crystal structure of OCP is constructed of compiled apatite layers and water layers, which make possible the incorporation of various ions in its structure, making it feasible to alter the overall effect OCP has in the system. Next to that intrinsic property, characteristics as high solubility, biodegradability and osteoconductivity have made it indispensable to tailor OCP as a carrier material. In this review, we present the main characteristics and progress done on utilizing OCP as an innovative vehicle and provide suggestions for possible research pathways and advantages for local drug delivery in bone tissue engineering. STATEMENT OF SIGNIFICANCE: Octacalcium phosphate (OCP), being a precursor to biologically formed apatite, has many assets when compared to other calcium phosphates. Owing to its highly pertinent structure, it is being used as a vehicle for biologically active substances or ions for bone regeneration. However, orchestrating drug delivery systems with OCP, in order to achieve the best possible outcome, is still a pioneering concept, and the all-encompassing data is still scarce. Although several articles have been published on this matter, to this date there is no systematic overview pointing out the benefits that OCP can bring in the field of drug delivery. Here we offer a comprehensive overview, starting from the OCP synthesis to its structure, morphology, and the biological significance OCP has.

The fate of biomaterials is orchestrated by biocompatibility and bioregulation characteristics, reported to be closely related to topographical structures. For the purpose to investigate the topography of fibrous membranes on the guided bone regeneration performance, we successfully fabricated poly (lactate-co-glycolate)/fish collagen/nano-hydroxyapatite (PFCH) fibrous membranes with random, aligned and latticed topography by electrospinning. The physical, chemical and biological properties of the three topographical PFCH membranes were systematically investigated by in vitro and in vivo experiments. The subcutaneous implantation of C57BL6 mice showed an acceptable mild foreign body reaction of all three topological membranes. Interestingly, the latticed PFCH membrane exhibited superior abilities to recruit macrophage/monocyte and induce angiogenesis. We further investigated the osteogenesis of the three topographical PFCH membranes via the critical-size calvarial bone defect model of rats and mice and the results suggested that latticed PFCH membrane manifested promising performance to promote angiogenesis through upregulation of the HIF-1α signaling pathway; thereby enhancing bone regeneration. Our research illustrated that the topological structure of fibrous membranes, as one of the characteristics of biomaterials, could regulate its biological functions, and the fibrous structure of latticed topography could serve as a favorable surface design of biomaterials for bone regeneration. Statement of significance In material-mediated regeneration medicine, the interaction between the biomaterial and the host is key to successful tissue regeneration. The micro-and nano-structure becomes one of the most critical physical clues for designing biomaterials. In this study, we fabricated three topological electrospun membranes (Random, Aligned and Latticed) to understand how topological structural clues mediate bone tissue regeneration. Interestingly, we found that the Latticed topographical PFCH membrane promotes macrophage recruitment, angiogenesis, and osteogenesis in vivo, indicating the fibrous structure of latticed topography could serve as a favorable surface design of biomaterials for bone regeneration.

Titanium has been used in various biomedical applications; however, titanium exhibits poor wear resistance, and its bioinert surface slows osseointegration in vivo. In this study, directed energy deposition (DED)-based additive manufacturing (AM) was used to process hydroxyapatite (HA) reinforced Ti6Al4V (Ti64) composites to improve biocompatibility and wear resistance simultaneously. Electron micrographs of the composites revealed dense microstructures where HA is observed at the β-phase grain boundaries. Hardness was observed to increase by 57% and 71% for 2 and 3 wt.% HA in Ti64 composites, respectively. XRD analysis revealed no change in the present phases. Tribological studies revealed an increase in contact resistance due to in situ HA-based tribofilm formation, reduction in wear rate when testing in DMEM with a ZrO(2) counter wear ball

Hydroxyapatite (HA) nanorods in the collagen matrix of bone have a macroscopically ordered structure that has many similarities to the ordered structure of anisotropic nano-units in inorganic liquid crystals (LCs). Inspired by these similarities, we conducted the first (to our best knowledge) synthesis of HA LCs in non-polar solvents (such as cyclohexane and toluene), thus expanding the range of applicable monomers and polymers. We synthesized HA nanorods by a simple, effective, and oleic-acid-assisted hydrothermal route. The hydrothermal temperature directly modulates the aspect ratio of the HA nanorods, and indirectly modulates their LC behavior. The LC phase transition has no size limitation. Thus, our approach may be used to develop high solid content, macroscopically assembled, large-scale polymer-based bio(mimetic)-materials.

In this study a high resolution structural analysis revealed that enamel prisms are surrounded by an interface that is discontinuous with frequent mineral to mineral contact separated by gaps. This contact manifests either by crystallites bridging the boundary between prismatic and interprismatic enamel or continuous crystallites curving and bridging the interprismatic enamel to the prisms. The geometrical resolution of this TEM investigation of the interfaces is ≤2 nm as a basis for micromechanical models. Within this resolution, contrary to existing structural descriptions of dental enamel structure in materials science literature, here the crystallites themselves are shown to be either in direct contact with each other, sometimes even fusing together, or are separated by gaps. Image analysis revealed that on average only 57 ± 15% of the interface consists of points of no contact between crystallites. This work reveals structural features of dental enamel that contribute important understanding to both the architecture and mechanical properties of this biological material. A new structural model is proposed and the implications for the mechanical properties of dental enamel are discussed. Statement of significance In this study a high resolution structural analysis, employing focused ion beam and transmission electron microscopy revealed that enamel prisms are surrounded by interfaces that are discontinuous with frequent mineral to mineral contact separated by gaps. Although the interfaces in enamel have been investigated previously, existing studies are lacking in detail considering the geometry and morphology of the interfaces. We think that this result is of great importance when it comes to the understanding of the mechanical properties. In our opinion the concept of soft sheaths is no longer feasible. The resulting observations are included in a new structural model which provides new qualitative insights into the mechanical behavior. Existing analytical models were applied to simulate the new geometrical structure.

Nanostructured hydroxyapatite (HAp) has been applied widely as a scaffold material for bone tissue engineering for its good osteoinduction and biodegradability. However, the degradation process and the distribution of degraded HAp within the bone-defect cavity is still not clear. To visually study the behavior of HAp in bone repair process, a membrane of HAp/terbium (Tb)-HAp nanowires (NWs) was prepared with a concentric circle structure (CCS), of which the inner circle and the outer ring were constructed with Tb-HAp and HAp NWs, respectively. HAp/Tb-HAp CCS membrane possessed good osteogenic capacity and efficient fluorescence in the center for visualization. The in vitro experimental results proved that the Tb-HAp and HAp NWs membranes both presented high cytocompatibility and adequate efficiency to induce osteogenic differentiation of bone marrow stem cells (BMSCs). HAp/Tb-HAp CCS membranes were then implanted into a rat calvarial bone-defect model to study the behavior of HAp in bone repair process in vivo by tracking the fluorescence distribution. The results showed that the fluorescence of Tb-HAp diffused gradually from the inner circle to the outer ring, which suggested that the HAp was first degraded, and then the degraded product was diffused and finally reconstructed. Further, the histological results proved that the doping of Tb did not impair the promotive effect of HAp on bone repair process. Therefore, this study provided a visual method to observe the degradation-diffusion-reconstruction behavior of HAp nanomaterials in bone repair process. Statement of significance The study of dynamic degradation process of implanted hydroxyapatite (HAp) materials in bone-defect cavity is of great significance to bone tissue engineering applications. Here, we designed a HAp/Tb-HAp nanowires (NWs) membrane with concentric circle structure (CCS) to visibly observe the behavior of HAp during bone repair process. HAp/Tb-HAp CCS membrane possessed both osteoinduction ability and fluorescence property. Calvarial bone-defect repair experiments in vivo showed that the fluorescence of Tb-HAp diffused gradually from inner circle to outer ring, which suggested that HAp was first degraded, then diffused and finally reconstructed. Therefore, this invention provides not only a visible method to observe the degradation-diffusion-reconstruction behavior of HAp-based biomaterials, but also a basic understanding of the dynamic change of HAp-based biomaterials.

Infection is a main cause of implant failure. Early implant-related infections often occur in the first 4 weeks post-operation. Inhibiting bacterial adhesion and biofilm formation at the early stage and promoting subsequent implant osseointegration are important for implant success. Our previous studies demonstrated that dimethylaminododecyl methacrylate (DMADDM) provided dental materials with antibacterial effects. In the present study, DMADDM and hydroxyapatite (HA) are loaded on to the titanium (Ti) surface via poly dopamine (PDA) self-polymerization. This local DMADDM-delivery Ti is referred as Ti-PHD. Here we report the two-staged capability of Ti-PHD: (1) in the first stage, releasing DMADDM during the high-infection-risk initial period post-implantation for 4 weeks; (2) then in the second stage, enhancing osteogenesis and promoting osseointegration. Ti-PHD has a porous surface with higher average roughness and greater hydrophilicity than pure Ti. Its biocompatibility is verified in vitro and in vivo. During the first 4 weeks of release, both DMADDM remaining on Ti surface and DMADDM released into the soaking medium greatly reduced the adherence and growth of pathogens. This is further confirmed by the prevention of bone destruction in a rat osteomyelitis model. After releasing DMADDM for 4 weeks, Ti-PHD promotes osteogenic differentiation of human bone marrow mesenchymal stem cells (hBMSCs) and new bone formation around the implants in vivo. This article represents the first report on the two-staged, time-dependent antibacterial and osteogenesis effects of Ti-PHD, demonstrating its potential for clinical applications to inhibit implant-associated infections. STATEMENT OF SIGNIFICANCE: The present study develops a two-staged time-dependent system for local dimethylaminododecyl methacrylate (DMADDM) delivery via Ti implant (referred to as Ti-PHD). DMADDM and hydroxyapatite (HA) are loaded on to the Ti surface with poly dopamine (PDA). Ti-PHD can release DMADDM during the high-risk period of infection in the first stage, and then promote osseointegration and new bone formation in the second stage. This bioactive and therapeutic Ti is promising to inhibit infections and enhance implant success.

In this study, we developed aptamer-conjugated hydroxyapatite (Apt-HA) that promotes bone regeneration and angiogenesis. The 3R02 bivalent aptamer specific to vascular endothelial growth factor (VEGF) was grafted to the hydroxyapatite (HA) surface. Apt-HA was tested for its VEGF protein capture ability to determine the optimal aptamer concentration immobilized on the HA. Apt-HA showed higher VEGF protein capture ability, and faster growth of human umbilical vein endothelial cell (HUVEC) compared to a neat HA with no cytotoxic effects on human osteoblasts. To examine in vivo angiogenesis and bone regeneration, Apt-HA and HA were bilaterally implanted into rabbit tibial metaphyseal defects and analyzed after eight weeks using micro-CT, histology, and histomorphometry. Apt-HA showed significantly increased the volume of new bones, the percentage of bone, and the density of bone mineral in cortical bone. Apt-HA also exhibited the enhanced bone formation at the cortical region in a histomorphometric analysis. Finally, Apt-HA showed significantly increased blood vessel number compared to a neat HA. In summary, the engineered Apt-HA has potential as a bone graft material that may simultaneously promote bone regeneration and angiogenesis. STATEMENT OF SIGNIFICANCE: This work presents a functional hydroxyapatite bone graft using a DNA-based aptamer which overcomes the limitations of existing bone graft materials, which use bound signaling peptides. DNA aptamer immobilized hydroxyapatite enhances the in vitro proliferation of human umbilical vascular endothelial cells as well as in vivo angiogenesis and bone regeneration. DNA aptamer immobilized hydroxyapatite shows no cytotoxic effect on human osteoblasts.

The design and development of bioactive materials that are inherently conducive for osteointegration and bone regeneration with tunable mechanical properties and degradation remains a challenge. Herein, we report the development of a new class of citrate-based materials with glycerophosphate salts, β-glycerophosphate disodium (β-GP-Na) and glycerophosphate calcium (GP-Ca), incorporated through a simple and convenient one-pot condensation reaction, which might address the above challenge in the search of suitable orthopedic biomaterials. Tensile strength of the resultant poly (octamethylene citrate glycerophosphate), POC-βGP-Na and POC-GP-Ca, was as high as 28.2 ± 2.44 MPa and 22.76 ± 1.06 MPa, respectively. The initial modulus ranged from 5.28 ± 0.56 MPa to 256.44 ± 22.88 MPa. The mechanical properties and degradation rate of POC-GP could be controlled by varying the type of salts, and the feeding ratio of salts introduced. Particularly, POC-GP-Ca demonstrated better cytocompatibility and the corresponding composite POC-GP-Ca/hydroxyapatite (HA) also elicited improved osteogenic differentiation of human mesenchymal stem cells (hMSCs) in vitro, as compared to POC-βGP-Na/HA and POC/HA. The superior in-vivo performance of POC-GP-Ca/HA microparticle scaffolds in promoting bone regeneration over POC-βGP-Na/HA and POC/HA was further confirmed in a rabbit femoral condyle defect model. Taken together, the tunability of mechanical properties and degradation rates, together with the osteopromotive nature of POC-GP polymers make these materials, especially POC-GP-Ca well suited for bone tissue engineering applications. Statement of Significance The design and development of bioactive materials that are inherently conducive for osteointegration and bone regeneration with tunable mechanical properties and degradation remains a challenge. Herein, we report the development of a new class of citrate-based materials with glycerophosphate salts, β-glycerophosphate disodium (β-GPNa) and glycerophosphate calcium (GPCa), incorporated through a simple and convenient one-pot condensation reaction. The resultant POC-GP polymers showed significantly improved mechanical property and tunable degradation rate. Within the formulation investigated, POC-GPCa/HA composite further demonstrated better bioactivity in favoring osteogenic differentiation of hMSCs in vitro and promoted bone regeneration in rabbit femoral condyle defects. The development of POC-GP expands the repertoire of the well-recognized citrate-based biomaterials to meet the ever-increasing needs for functional biomaterials in tissue engineering and other biomedical applications.

In this study, the potential of vascularized tissue-engineered bone constructed by a double cell-sheet (DCS) complex and polylysine (PLL)-modified coralline hydroxyapatite (CHA) to repair large radius bone defects was investigated in rabbits. Firstly, the DCS complex was obtained after rabbit adipose-derived mesenchymal stem cell (ADSC) culture was induced. Secondly, PLL-CHA composite scaffolds with different concentrations of PLL were prepared by the soaking and vacuum freeze-drying methods, and then the scaffolds were characterized by X-ray diffraction (XRD), Fourier transform infrared (FTIR) spectroscopy, compression performance testing and cytocompatibility evaluation. Thirdly, DCS-PLL-CHA vascularized tissue-engineered bone was constructed in vitro and transplanted into a large radius bone defect model in rabbits. Finally, the potential of the DCS-PLL-CHA vascularized tissue-engineered bone to repair the large bone defect was evaluated through general observations, laser speckle imaging, scanning electron microscopy (SEM), histological staining, radiography observations and RT-PCR. The in vitro experimental results showed that the DCS complex provided a very large cell reserve, which carried a large number of osteoblasts and vascular endothelial cells that were induced in vitro. When the DCS complex was combined with the PLL-CHA scaffold in vitro, the effects of PLL on cell adhesion, proliferation and differentiation led to a situation similar to the chemotaxis of the body, making the combined complex more conducive to graft cellularization than the DCS complex alone. The in vivo experiments showed blood supply on the surface of the callus in each group, and the amount of blood perfusion on the surface of the defect area was almost equal among the groups. At 12 weeks, the surface of the DCS-PLL-CHA group was completely wrapped by bone tissue and osteoids, the cortical bone image was basically continuous, and the medullary cavity was mainly perforated. A large amount of well-arranged lamellar bone was formed, a small amount of undegraded CHA exhibited a linear pattern, and a large amount of bone filling could be seen in the pores. At 12 weeks, the expression levels of BGLAP, SPP1 and VEGF were similar in each group, but PECAM1 expression was higher in the DCS-PLL-CHA group than in the autogenous bone group and CHA group. The results showed that PLL could effectively promote the adhesion, proliferation and differentiation of ADSCs and that DCS-PLL-CHA vascularized tissue-engineered bone has potential for bone regeneration and bone reconstruction and can be used to repair large bone defects. Statement of Significance 1. PLL-CHA composite scaffolds with different concentrations of PLL were prepared by the soaking and vacuum freeze-drying methods. 2. The vascularized tissue-engineered bone was constructed by the double cell sheet (DCS) complex combined with PLL-CHA scaffolds. 3. The DCS-PLL-CHA vascularized tissue-engineered bone has potential for bone regeneration and bone reconstruction and can be used to repair large bone defects.

Recent developments in 3D printing (3DP) research have led to a variety of scaffold designs and techniques for osteochondral tissue engineering; however, the simultaneous incorporation of multiple types of gradients within the same construct remains a challenge. Herein, we describe the fabrication and mechanical characterization of porous poly(e-caprolactone) (PCL) and PCL-hydroxyapatite (HA) scaffolds with incorporated vertical porosity and ceramic content gradients via a multimaterial extrusion 3DP system. Scaffolds of 0 wt% HA (PCL), 15 wt% HA (HA15), or 30 wt% HA (HA30) were fabricated with uniform composition and porosity (using 0.2 mm, 0.5 mm, or 0.9 mm on-center fiber spacing), uniform composition and gradient porosity, and gradient composition (PCL-HA15-HA30) and porosity. Micro-CT imaging and porosity analysis demonstrated the ability to incorporate both vertical porosity and pore size gradients and a ceramic gradient, which collectively recapitulate gradients found in native osteochondral tissues. Uniaxial compression testing demonstrated an inverse relationship between porosity, ϕ, and compressive modulus, E, and yield stress, σy, for uniform porosity scaffolds, however, no differences were observed as a result of ceramic incorporation. All scaffolds demonstrated compressive moduli within the appropriate range for trabecular bone, with average moduli between 86 ± 14–220 ± 26 MPa. Uniform porosity and pore size scaffolds for all ceramic levels had compressive moduli between 205 ± 37–220 ± 26 MPa, 112 ± 13–118 ± 23 MPa, and 86 ± 14–97 ± 8 MPa respectively for porosities ranging between 14 ± 4–20 ± 6%, 36 ± 3–43 ± 4%, and 54 ± 2–57 ± 2%, with the moduli and yield stresses of low porosity scaffolds being significantly greater (p 0.05) to those of the highest porosity uniform scaffolds (porosity gradient scaffolds 98 ± 23–107 ± 6 MPa, and 102 ± 7 MPa for dual composition/porosity gradient scaffolds), indicating that these properties are more heavily influenced by the weakest section of the gradient. The compression data for uniform scaffolds were also readily modeled, yielding scaling laws of the form E ∼ (1 − ϕ)1.27 and σy ∼ (1 − ϕ)1.37, which demonstrated that the compressive properties evaluated in this study were well-aligned with expectations from previous literature and were readily modeled with good fidelity independent of polymer scaffold geometry and ceramic content. All uniform scaffolds were similarly deformed and recovered despite different porosities, while the large-pore sections of porosity gradient scaffolds were significantly more deformed than all other groups, indicating that porosity may not be an independent factor in determining strain recovery. Moving forward, the technique described here will serve as the template for more complex multimaterial constructs with bioactive cues that better match native tissue physiology and promote tissue regeneration. Statement of significance This manuscript describes the fabrication and mechanical characterization of “dual” porosity/ceramic content gradient scaffolds produced via a multimaterial extrusion 3D printing system for osteochondral tissue engineering. Such scaffolds are designed to better address the simultaneous gradients in architecture and mineralization found in native osteochondral tissue. The results of this study demonstrate that this technique may serve as a template for future advances in 3D printing technology that may better address the inherent complexity in such heterogeneous tissues.

The design of hydrogels with adequate mechanical properties and excellent bioactivity, osteoconductivity, and capacity for osseointegration is essential to bone repair and regeneration. However, it is challenging to integrate all these properties into one bone scaffold. Herein, we developed a strong, tough, osteoconductive hydrogel by a facile one-step micellar copolymerization of acrylamide and urethacrylate dextran (Dex-U), followed by the in situ mineralization of hydroxyapatite (HAp) nanocrystals. We show that the soft, flexible, and hydrophobically associated polyacrylamide (PAAm) network is strengthened by the stiff crosslinked Dex-U phase, and that the mineralized HAp simultaneously improves the mechanical properties and osteoconductivity. The obtained HAp mineralized PAAm/Dex-U hydrogel (HAp-PADH) has extremely high compressive strength (6.5 MPa) and enhanced fracture resistance (over 2300 J m−2), as compared with pure PAAm hydrogels. In vitro, we show that the mineralized HAp layer promotes the adhesion and proliferation of osteoblasts, and effectively stimulates osteogenic differentiation. Through the in vivo evaluation of hydrogels in a femoral condyle defect rabbit model, we show regeneration of a highly mineralized bone tissue and direct bonding to the HAp-PADH interface. These findings confirm the excellent osteoconductivity and osseointegration ability of fabricated HAp-PADH. The present HAp-PADH, with its superior mechanical properties and excellent osteoconductivity, should have great potential for bone repair and regeneration. Statement of Significance We developed a strong, tough, and osteoconductive hydrogel by a facile one-step micellar copolymerization of acrylamide and urethane methacrylate dextran (Dex-U), followed by the in situ mineralization of hydroxyapatite (HAp) nanocrystals. The hydrophobic micellar copolymerization and introduction of the stiff crosslinked Dex-U phase endowed the soft polyacrylamide (PAAm) network with enhanced strength and toughness. The in situ mineralized HAp nanocrystals on the hydrogels further improved the mechanical properties of the hydrogels and promoted osteogenic differentiation of cells. Mechanical tests together with in vitro and in vivo evaluations confirmed that the HAp mineralized PAAm/Dex-U hydrogel (HAp-PADH) achieved a combination of superior mechanical properties and excellent osseointegration, and thus may offer a promising candidate for bone repair and regeneration.

Coatings based on ion-substituted calcium phosphate (Ca-P) have attracted great attention in the scientific community over the past decade for the development of biomedical applications. Among such Ca-P based structures, hydroxyapatite (HA) has shown significant influence on cell behaviors including cell proliferation, adhesion, and differentiation. These cell behaviors determine the osseointegration between the implant and host bone and the biocompatibility of implants. This review presents a critical analysis on the physical vapor deposition magnetron sputtering (PVDMS) technique that has been used for ion-substituted Ca-P based coatings on implants materials. The effect of PVDMS processing parameters such as discharge power, bias voltage, deposition time, substrate temperature, and post-heat treatment on the surface properties of ion-substituted Ca-P coatings is elucidated. Moreover, the advantages, short comings and future research directions of Ca-P coatings by PVDMS have been comprehensively analyzed. It is revealed that the topography and surface chemistry of amorphous HA coatings influence the cell behavior, and ion-substituted HA coatings significantly increase cell attachment but may result in a cytotoxic effect that reduces the growth of the cells attached to the coating surface areas. Meanwhile, low-crystalline HA coatings exhibit lower rates of osteogenic cell proliferation as compared to highly crystalline HA coatings developed on Ti based surfaces. PVDMS allows a close reproduction of bioapatite characteristics with high adhesion strength and substitution of therapeutic ions. It can also be used for processing nanostructured Ca-P coatings on polymeric biomaterials and biodegradable metals and alloys with enhanced corrosion resistance and biocompatibility. STATEMENT OF SIGNIFICANCE: Recent studies have utilized the physical vapor deposition magnetron sputtering (PVDMS) for the deposition of Ca-P and ion-substituted Ca-P thin film coatings on orthopedic and dental implants. This review explains the effect of PVDMS processing parameters, such as discharge power, bias voltage, deposition time, substrate temperature, and post-heat treatment, on the surface morphology and crystal structure of ion-substituted Ca-P and ion-substituted Ca-P thin coatings. It is revealed that coating thickness, surface morphology and crystal structure of ion-substituted Ca-P coatings via PVDMS directly affect the biocompatibility and cell responses of such structures. The cell responses determine the osseointegration between the implant and host bone and eventually the success of the implants.

Emerging studies indicate hydroxyapatite nanoparticles (HANPs) exhibit modest immunogenicity to elicit innate immune response which might involve Toll-like receptor 4 (TLR4) activation. This study was proposed to elucidate how HANPs direct over TLR4 signal activity in macrophage in response to TLR4 ligand, lipopolysaccharide (LPS). The present study for the first time reveals that HANPs themselves can induce TLR4 endocytosis and activate pathways both of nuclear factor-kappa B (NF-κB) and interferon regulatory factor 3 (IRF3), which potentially trigger the production of inflammatory cytokine by macrophage. Further, HANPs dose-dependently reprogram over LPS driven TLR4 signaling transduction in macrophage, leading to synergistically augmented innate immune response. In particular, HANPs synergize with LPS to promote macrophage polarization toward M1 phenotype. Moreover, HANPs abrogate the endotoxin tolerance in macrophages by restoring the production of inflammatory cytokines from macrophage in response to secondary LPS stimulation, and enhance the responsiveness of the body to LPS re-challenge in the endotoxin tolerance mice model. Therefore, this study sheds a new light on the mechanism by which HANPs drive the innate immune response, and offers a powerful strategy to potentiate LPS mediated TLR4 signaling activation in macrophage. Statement of significance In recent years, increasing attention has been given to hydroxyapatite nanoparticles (HANPs) on how they interact with immune cells for achieving appropriate biological effect such as bone tissue repair, soft tissue filler, tumor treatment, vaccine delivery, et al. This study indicated HANPs can induce TLR4 signaling activation. In the further, HANPs dose-dependently synergize with LPS to program over LPS induced TLR4 signaling transduction in macrophage, to favor macrophage polarizing toward M1 phenotype, as well as to abrogate immune tolerance in macrophage in response to repeated LPS stimulation. This work opens a window for the intrinsic mechanism of HANPs to drive immune response and facilitate to direct the rational use or design of HANPs for their better biomedical application.

Doxorubicin (DOX) is a cornerstone drug in the treatment of osteosarcoma. However, achieving sufficient concentration in the tumor tissue after systemic administration with few side effects has been a challenge. Even with the most advanced nanotechnology approaches, less than 5% of the total administered drug gets delivered to the target site. Alternatives to increase the local concentration of DOX within the tumor using improved drug delivery methods are needed. In this study, we evaluate a clinically approved calcium sulfate/hydroxyapatite (CaS/HA) carrier, both in-vitro and in-vivo, for local, sustained and controlled delivery of DOX to improve osteosarcoma treatment. In-vitro drug release studies indicated that nearly 28% and 36% of the loaded drug was released over a period of 4-weeks at physiological pH (7.4) and acidic pH (5), respectively. About 63% of the drug had been released after 4-weeks in-vivo. The efficacy of the released drug from the CaS/HA material was verified on two human osteosarcoma cell lines MG-63 and 143B. It was demonstrated that the released drug fractions functioned the same way as the free drug without impacting its efficacy. Finally, the carrier system with DOX was assessed using two clinically relevant human osteosarcoma xenograft models. Compared to no treatment or the clinical standard of care with systemic DOX administration, the delivery of DOX using a CaS/HA biomaterial could significantly hinder tumor progression by inhibiting angiogenesis and cell proliferation. Our results indicate that a clinically approved CaS/HA biomaterial containing cytostatics could potentially be used for the local treatment of osteosarcoma. STATEMENT OF SIGNIFICANCE: The triad of doxorubicin (DOX), methotrexate and cisplatin has routinely been used for the treatment of osteosarcoma. These drugs dramatically improved the prognosis, but 45-55% of the patients respond poorly to the treatment with low 5-year survival. In the present study, we repurpose the cornerstone drug DOX by embedding it in a calcium sulfate/hydroxyapatite (CaS/HA) biomaterial, ensuring a spatio-temporal drug release and a hypothetically higher and longer lasting intra-tumoral concentration of DOX. This delivery system could dramatically hinder the progression of a highly aggressive osteosarcoma compared to systemic administration, by inhibiting angiogenesis and cell proliferation. Our data show an efficient method for supplementary osteosarcoma treatment with possible rapid translational potential due to clinically approved constituents.

Porosity plays a key role on the osteogenic performance of bone scaffolds. Direct Ink Writing (DIW) allows the design of customized synthetic bone grafts with patient-specific architecture and controlled macroporosity. Being an extrusion-based technique, the scaffolds obtained are formed by arrays of cylindrical filaments, and therefore have convex surfaces. This may represent a serious limitation, as the role of surface curvature and more specifically the stimulating role of concave surfaces in osteoinduction and bone growth has been recently highlighted. Hence the need to design strategies that allow the introduction of concave pores in DIW scaffolds. In the current study, we propose to add gelatin microspheres as a sacrificial material in a self-setting calcium phosphate ink. Neither the phase transformation responsible for the hardening of the scaffold nor the formation of characteristic network of needle-like hydroxyapatite crystals was affected by the addition of gelatin microspheres. The partial dissolution of the gelatin resulted in the creation of spherical pores throughout the filaments and exposed on the surface, increasing filament porosity from 0.2 % to 67.9 %. Moreover, the presence of retained gelatin proved to have a significant effect on the mechanical properties, reducing the strength but simultaneously giving the scaffolds an elastic behavior, despite the high content of ceramic as a continuous phase. Notwithstanding the inherent difficulty of in vitro cultures with this highly reactive material an enhancement of MG-63 cell proliferation, as well as better spreading of hMSCs was recorded on the developed scaffolds. STATEMENT OF SIGNIFICANCE: Recent studies have stressed the role that concave surfaces play in tissue regeneration and, more specifically, in osteoinduction and osteogenesis. Direct ink writing enables the production of patient-specific bone grafts with controlled architecture. However, besides many advantages, it has the serious limitation that the surfaces obtained are convex. In this article, for the first time we develop a strategy to introduce concave pores in the printed filaments of biomimetic hydroxyapatite by incorporation and partial dissolution of gelatin microspheres. The retention of part of the gelatin results in a more elastic behavior compared to the brittleness of hydroxyapatite scaffolds, while the needle-shaped nanostructure of biomimetic hydroxyapatite is maintained and gelatin-coated concave pores on the surface of the filaments enhance cell spreading.

Most fractures heal by a combination of endochondral and intramembranous ossification dependent upon strain and vascularity at the fracture site. Many biomaterials-based bone regeneration strategies rely on the use of calcium phosphates such as nano-crystalline hydroxyapatite (nHA) to create bone-like scaffolds. In this study, nHA was dispersed in reactive polymers to form composite scaffolds that were evaluated both in vitro and in vivo. Matrix assays, immunofluorescent staining, and Western blots demonstrated that nHA influenced mineralization and subsequent osteogenesis in a dose-dependent manner in vitro. Furthermore, nHA dispersed in polymeric composites promoted osteogenesis by a similar mechanism as particulated nHA. Scaffolds were implanted into a 2-mm defect in the femoral diaphysis or metaphysis of Sprague-Dawley rats to evaluate new bone formation at 4 and 8 weeks. Two formulations were tested: a poly(thioketal urethane) scaffold without nHA (PTKUR) and a PTKUR scaffold augmented with 22 wt% nHA (22nHA). The scaffolds supported new bone formation in both anatomic sites. In the metaphysis, augmentation of scaffolds with nHA promoted an intramembranous healing response. Within the diaphysis, nHA inhibited endochondral ossification. Immunohistochemistry was performed on cryo-sections of the bone/scaffold interface in which CD146, CD31, Endomucin, CD68, and Myeloperoxidase were evaluated. No significant differences in the infiltrating cell populations were observed. These findings suggest that nHA dispersed in polymeric composites induces osteogenic differentiation of adherent endogenous cells, which has skeletal site-specific effects on fracture healing. STATEMENT OF SIGNIFICANCE: Understanding the mechanism by which synthetic scaffolds promote new bone formation in preclinical models is crucial for bone regeneration applications in the clinic where complex fracture cases are seen. In this study, we found that dispersion of nHA in polymeric scaffolds promoted in vitro osteogenesis in a dose-dependent manner through activation of the PiT1 receptor and subsequent downstream Erk1/2 signaling. While augmentation of polymeric scaffolds with nHA enhanced intramembranous ossification in metaphyseal defects, it inhibited endochondral ossification in diaphyseal defects. Thus, our findings provide new insights into designing synthetic bone grafts that complement the skeletal site-specific fracture healing response.

The phase transformation from soluble calcium phosphates to less-soluble hydroxyapatite (HAP) is a thermodynamically natural route. This process is irreversible, and effective use of poorly reactive HAP to repair teeth that have no cellular metabolism remains challenging. However, this thermodynamically controlled transformation may apparently be reversed through the fast nucleation and growth of metastable phases, leading to a reactive HAP surface. Here, the assembled HAP-nanorod phase is demonstrated to change into the metastable octacalcium phosphate (OCP) phase in a calcium phosphate solution containing 0.8 ppm fluoride. Grown OCPs display parallel surface streaks and their 1 1 ¯ 0 and 00l (l: odd) electron-diffraction spots are often not visible. The streaked, elongated OCP gradually grows into large plates with flat surfaces that exhibit an intense 1 1 ¯ 0 spot. Crystal-structure models reveal that the unique epitaxial overgrowth of OCP on HAP occurs since both materials share coherent {100} faces, resulting in the distinctive disappearance of 1 1 ¯ 0 and 00l OCP spots. A polysynthetic twin model that reliably explains this disappearance is proposed for the growth of OCP. This apparent reverse phase transformation produces hybrid calcium phosphates consisting of HAP cores and highly reactive outer OCP layers that are promising for the repair of dentin caries. Statement of significance This paper demonstrates important and interesting finding regarding formation of calcium phosphates in relation to their crystal structures. We first show that hydroxyapatite (HAP), the major constituent of human teeth and bone, can reversely change to its precursor, octacalcium phosphate (OCP), contrary to thermodynamic-stability rule. This apparent reverse phase transformation occurs through sharing the coherent {100} faces of both materials under controlled fluoride concentration. Nanoscale similarity of two crystal surfaces enables structurally shared epitaxial overgrowth of OCP on HAP aided by faster growth rate of OCP than that of HAP. This reaction produces hybrid crystal consisting of outer OCP and core HAP, that has not been known before and is able to be applied to dentin caries repair.

Amorphous Ca-phosphate (ACP) particles stabilized by inorganic polyphosphate (polyP) were prepared by co-precipitation of calcium and phosphate in the presence of polyP (15% [w/w]). These hybrid nanoparticles showed no signs of crystallinity according to X-ray diffraction analysis, in contrast to the particles obtained at a lower (5% [w/w]) polyP concentration or to hydroxyapatite. The ACP/15% polyP particles proved to be a suitable matrix for cell growth and attachment and showed pronounced osteoblastic and vasculogenic activity in vitro. They strongly stimulated mineralization of the human osteosarcoma cell line SaOS-2, as well as cell migration/microvascularization, as demonstrated in the scratch assay and the in vitro angiogenesis tube forming assay. The possible involvement of an ATP gradient, generated by polyP during tube formation of human umbilical vein endothelial cells, was confirmed by ATP-depletion experiments. In order to assess the morphogenetic activity of the hybrid particles in vivo, experiments in rabbits using the calvarial bone defect model were performed. The particles were encapsulated in poly(d,l-lactide-co-glycolide) microspheres. In contrast, to crystalline Ca-phosphate (containing only 5% [w/w] polyP) or to crystalline β-tricalcium phosphate, amorphous ACP/15% polyP particles caused pronounced osteoinductive activity already after a six-week healing period. The synthesis of new bone tissue was accompanied by an intense vascularization and an increased expression of mineralization/vascularization marker genes. The data show that amorphous polyP-stabilized ACP, which combines osteoinductive activity with the ability to act as a precursor of hydroxyapatite formation both in vitro and in vivo, is a promising material for bone regeneration.

International audience; A better understanding of bone nanostructure around the bone-implant interface is essential to improve longevity of clinical implants and decrease failure risks. This study investigates the spatio-temporal evolution of mineral crystal thickness and plate orientation in newly formed bone around the surface of a metallic implant. Standardized coin-shaped titanium implants designed with a bone chamber were inserted into rabbit tibiae for 7 and 13 weeks. Scanning measurements with micro-focused small-angle X-ray scattering (SAXS) were carried out on newly formed bone close to the implant and in control mature cortical bone. Mineral crystals were thinner close to the implant (1.8 ± 0.45 nm at 7 weeks and 2.4 ± 0.57 nm at 13 weeks) than in the control mature bone tissue (2.5 ± 0.21 nm at 7 weeks and 2.8 ± 0.35 nm at 13 weeks), with increasing thickness over healing time (+30 % in 6 weeks). These results are explained by younger bone close to the implant, which matures during osseointegration. Thinner mineral crystals parallel to the implant surface within the first 100 µm close to the implant indicate that the implant affects bone ultrastructure close to the implant, potentially due to heterogeneous interfacial stresses, and suggest a longer maturation process of bone tissue and difficulty in binding to the metal. The bone growth kinetics within the bone chamber was derived from the spatio-temporal evolution of bone tissue's nanostructure, coupled with microtomographic imaging. The findings indicate that understanding mineral crystal thickness or plate orientation can improve our knowledge of osseointegration.

Hydroxyapatite (HA) is the main inorganic component of human bone. Inspired by nacre and cortical bone, hydroxyapatite-based coil scaffolds were successfully prepared. The scaffolds presented "brick and mortar" multi-layered structure of nacre and multi-layered concentric circular structure of cortical bone. Because of bioactive components and hierarchical structure, the scaffolds possessed good compressive strength (≈95 MPa), flexural strength (≈161 MPa) and toughness (≈1.1 MJ/m3). In addition, they showed improved angiogenesis and osteogenesis in rat and rabbit critical sized bone defect models. By mimicking co-biological systems, this work provided a feasible strategy to optimize the properties of traditional tissue engineering biological materials for vascularized bone regeneration.

The surgical implant is an interdisciplinary therapeutic modality that offers unique advantages in the daily practice of otorhinolaryngology. Some well-known examples include cochlear implants, bone-anchored hearing aids, sinus stents, and tracheostomy tubes. Neuroprotective, osteogenic, anti-inflammatory, and antimicrobial effects are among their established or pursued functions. Implant-based drug delivery affords an efficient and potent approach to enhancing these therapeutic functions. Recent innovations have infiltrated all four elements of a drug-eluting implant. The purpose of this pre-clinical, biotechnology-oriented review is to discuss these developments in terms of the implant biomaterial, loaded medication, delivery pattern, and system fabrication. Cell-mediated neurotrophin release, fabrication of a hydroxyapatite-supported system, biodegradable polymer-based implants, and multiclass and multidrug delivery are some representative advancements. The ultimate goal here is to bridge the gap between biotechnology advances and clinical needs. The review is concluded with a perspective regarding the future opportunities and challenges in this popular and rapidly developing subject of research. STATEMENT OF SIGNIFICANCE: Surgical implants and local drug delivery are representative modern modalities of surgical treatment and medical treatment, respectively. Their synergy offers unique therapeutic advantages, such as minimal systemic side effects, proximity-related high efficiency, and potential absorbability. The applications of implant-based drug delivery have infiltrated otorhinolaryngology and head & neck surgery, which is well known for its related tissue diversity and surgical complexity. Examples discussed here include cochlear implants, bone-anchored hearing aids, sinus stents, and airway tubes. This timely review focuses primarily on the four fundamental components of an implant-based drug delivery system, namely implant biomaterial, loaded medication, delivery pattern, and system fabrication. A particular emphasis is placed upon the in vitro cellular and in vivo animal studies that demonstrate pre-clinical potentials.

Ideal artificial bone grafts aim for multiscale porosity, high mechanical strength and ensure rapid vascularization for bone ingrowth. In this work modular ceramic arteriovenous loops (AV-loops) with a hierarchical porosity approach were designed and manufactured to meet these criteria and to exceed the poor mechanical strength of monolithic scaffolds. Bioactive building blocks (β-TCP, HAp, BCP) with dimensions of 1.5–3.0 mm were prepared by injection molding and assembled to complex AV-loop scaffolds using a customized automated assembly technology (pick and place). The building blocks were bonded with a biocompatible adhesive. Single building blocks are characterized by a compressive strength of 112.4–134.5 MPa with a residual sintering porosity of 32.2–41.5%, matching the strength of cortical bone of 100–230 MPa. The compressive strength of the modular assemblies varied between 22.3 and 47.6 MPa primary depending on the building block arrangement. The achieved compressive strengths are superior to current monolithic AV-scaffolds and sufficient for the implantation as non-load-bearing AV-loop scaffolds in isolation chambers. The modular AV-loop scaffolds provide a hierarchical interconnected pore network (P = 58.8%) combining small macropores of 4.1–4.3 µm size for possible enhanced protein absorption and large gradient macropores of 200–1700 µm size for optimum vascularization and complete bone ingrowth. The modular building block approach allows to design patient individualized scaffolds with complex hierarchical pore networks. The pore volume, size and geometry as well as the biological response can effectively be tuned by changing the dimensions, shape and placing gap of the bioactive building blocks. Statement of Significance Gold standard of bone replacement in case of surgery or cancer is still own bone material usually taken from the hip/arm or leg in second surgery with poor mechanical properties and limited amount. To avoid a second surgery and provide mechanical strong scaffold structures for fast patient regeneration a novel modular building block approach is used. This allows complex scaffold geometry with a hierarchical interconnection porosity for blood vessel ingrowth. The pore volume, size and geometry as well as the biological response can effectively be tuned by changing the dimensions, shape and placing gap of the bioactive building blocks.

A double-cell sheet (DCS) complex composed of an osteogenic cell sheet and a vascular endothelial cell sheet with osteogenesis and blood vessel formation potential was developed in this study. The osteogenic and vascular endothelial cell sheets were obtained after induced culture of rabbit adipose-derived mesenchymal stem cells. The osteogenic cell sheet showed positive alizarin red, von Kossa, and alkaline phosphatase (ALP) staining. The vascular endothelial cell sheet exhibited visible W-P bodies in the cells, the expression of CD31 was positive, and a vascular mesh structure was spontaneously formed in a Matrigel matrix. The subcutaneous transplantation results for four groups of DCS and DCS-coral hydroxyapatite (CHA) complexes, and the CHA scaffold group in nude mice revealed mineralization of collagen fibers and vascularization in each group at 12 weeks, but the degrees of mineralization and vascularization showed differences among groups. The pattern involving endothelial cell sheets covered with osteogenic cell sheets, group B, exhibited the best results. In addition, the degree of mineralization of the DCS-CHA complexes was more mature than those of the same group of DCS complexes and the CHA scaffold, and the capillary number was greater than those of the same group of DCS complexes and the CHA scaffold. Therefore, the CHA scaffold strengthened the osteogenesis and blood vessel formation potential of the DCS complexes. Meanwhile, the DCS complexes also promoted the osteogenesis and blood vessel formation potential of the CHA scaffold. This study will provide a basis for building vascularized tissue-engineered bone for bone defect therapy. Statement of Significance This study developed a double-cell sheet (DCS) complex composed of an osteogenic cell sheet and a vascular endothelial cell sheet with osteogenesis and blood vessel formation potential. Osteogenic and vascular endothelial cell sheets were obtained after induced culture of rabbit adipose-derived mesenchymal stem cells. The DCS complex and DCS-CHA complex exhibited osteogenic and blood vessel formation potential in vivo. CHA enhanced the osteogenesis and blood vessel formation abilities of the DCS complexes in vivo. Meanwhile, the DCS complexes also promoted the osteogenesis and blood vessel formation potential of the CHA scaffold. Group B of the DCS complexes and DCS-CHA complexes exhibited the best osteogenesis and blood vessel formation abilities.

Since skin wounds are subject to bacterial infection and tissue regeneration may be impeded, there is demand for biomaterials that possess rapid bactericidal and tissue repair capability. Herein we report in situ promotion of wound healing by a photothermal therapy (PTT) assisted nanocatalytic antibacterial system utilizing a polydopamine (PDA) coating on hydroxyapatite (HAp) incorporated with gold nanoparticles (Au-HAp). The PDA@Au-HAp NPs produce hydroxyl radicals ( OH) via catalysis of a small concentration of H2O2 to render bacteria more vulnerable to the temperature change. The antibacterial efficacy against Escherichia coli and Staphylococcus aureus is 96.8% and 95.2%, respectively, at a controlled photo-induced temperature of 45 °C that causes no damage to normal tissues. By combining catalysis with near-infrared (NIR) photothermal therapy, the PDA@Au-HAp NPs provide safe, rapid, and effective antibacterial activity compared to OH or PTT alone. In addition, this system stimulates the tissue repairing-related gene expression to facilitate the formation of granulation tissues and collagen synthesis and thus accelerate wound healing. After the 10-day treatment of skin wounds in vivo, PDA@Au-HAp group exhibits quicker recovery than the control group and both sterilization and healing are completed after the 10-day treatment. Statement of significance This study presents in situ promotion of wound healing by a low-temperature photothermal therapy (PTT) assisted nanocatalytic antibacterial system utilizing a polydopamine (PDA) coating on hydroxyapatite (HAp) incorporated with gold nanoparticles (Au-HAp). The PDA@Au-HAp NPs produce hydroxyl radicals ( OH) via catalysis of a small concentration of H2O2 to render bacteria more vulnerable to temperature change. After irradiation by 808 nm laser, the antibacterial efficacy against Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) is 96.8% and 95.2%, respectively, at a low photo-induced temperature of 45 °C which causes no damage to normal tissues. In addition, this system stimulates the tissue repairing-related gene expression to facilitate the formation of granulation tissues and collagen synthesis and accelerate wound healing.

microRNAs offer vast therapeutic potential for multiple disciplines. From a bone perspective, inhibition of miR-133a may offer potential to enhance Runx2 activity and increase bone repair. This study aims to assess the therapeutic capability of antagomiR-133a delivery from collagen-nanohydroxyapatite (coll-nHA) scaffolds following cell-free implantation in rat calvarial defects (7 mm diameter). This is, to the best of our knowledge, the first report of successful in vivo antagomiR uptake in host cells of fully immunocompetent animals without distribution to other off-target tissues. Our results demonstrate the localized release of antagomiR-133a to the implant site at 1 week post-implantation with increased calcium deposits already evident in the antagomiR-133a loaded scaffolds at this early timepoint. This was followed by an approximate 2-fold increase in bone volume versus antagomiR-free scaffolds and a significant 10-fold increase over the empty defect controls, after just 4 weeks. An increase in host CD206

Recent studies indicate that there is a great demand to optimize pure Zn with tunable degradation rates and more desirable biocompatibility as orthopedic implants. Metal matrix composite (MMC) can be a promising approach for this purpose. In this study, MMC with pure Zn as a matrix and hydroxyapatite (HA) as reinforcements were prepared by spark plasma sintering (SPS). Feasibility of novel Zn-HA composites to be used as orthopedic implant applications was systematically evaluated. After sintering, HA distributed in the Zn particle boundaries uniformly. Corrosion tests indicated that the degradation rates of Zn-HA composites were adjustable due to the biphasic effects of HA. Zn-HA composites showed significantly improved cell viability of osteoblastic MC3T3-E1 cells compared with pure Zn. Both pure Zn and composites exhibited a low thrombosis risk and hemolysis rates while a Zn ion concentration-dependent effect was found on coagulation time. An effective antibacterial property was observed as well. The volume loss of pure Zn and Zn-5HA composite was 1.7% and 3.2% after 8 weeks’ implantation. Histological analysis found newly formed bone surrounding pure Zn and Zn-5HA composite at week 4 and increased bone mass over time. With prolonged implantation time, Zn-5HA composite was more effective on stimulating new bone formation than pure Zn. In summary, MMC is a feasible way to design Zn based materials with adjustable degradation rates and improved biocompatibility. Statement of Significance Biodegradable zinc materials are promising candidates for the new generation of orthopedic implants. However, the slow degradation rates and unsatisfactory cytocompatibility of pure Zn in bone environments limit its future clinical applications. Generally, alloying is a common way to improve the performance of pure Zn. In this study, metal matrix composite was chosen as a novel strategy to solve the problems. Hydroxyapatite, as a bioactive component, was added into Zn matrix via spark plasma sintering. We find that Zn-HA composites exhibited adjustable degradation rates and improved biocompatibility both in vitro and in vivo. This study provides exhaustive and significant information including microstructure, mechanical performance, degradation behavior, biocompatibility, hemocompatibility and antibacterial property for the future Zn based implants design.

Controllable osteoinduction maintained in the original defect area is the key to precise bone repair. To meet the requirement of precise bone regeneration, a hydroxyapatite (HAp) nanobelt/polylactic acid (PLA) (HAp/PLA) Janus membrane has been successfully prepared in this study by coating PLA on a paper-like HAp nanobelt film by a casting-pervaporation method. The Janus membrane possesses dual functions: excellent osteoinduction from the hydrophilic HAp nanobelt side and barrier function originating from the hydrophobic PLA film. The cell viability and osteogenic differentiation ability of human adipose-derived stem cells (hADSCs) on the Janus membrane were assessed. The in vitro experimental results prove that the HAp nanobelt side presents high cell viability and efficient osteoinduction without any growth factor and that the PLA side can prohibit cell attachment. The in vivo repair experiments on a rat mandible defect model prove that the PLA side can prevent postoperative adhesion between bone and adjacent soft tissues. Most importantly, the HAp side has a strong ability to promote defect repair and bone regeneration. Therefore, the HAp/PLA Janus membrane will have wide applications as a kind of tissue engineering material in precise bone repair because of its unique dual osteoinduction/barrier functions, biocompatibility, low cost, and its ability to be mass-produced. State of Significance Precise bone defect repair to keeping tissue integrity and original outline shape is a very important issue for tissue engineering. Here, we have designed and prepared a novel HAp/PLA Janus membrane using a casting-pervaporation method to form a layer of PLA film on paper-like HAp nanobelt film. HAp nanobelt side of the Janus membrane can successfully promote osteogenic differentiation. PLA side of the Janus membrane exhibits good properties as a barrier for preventing the adhesion of cells in vitro. Mandible repair experiments in vivo have shown that the HAp/PLA Janus membrane can promote rat mandible repair on the HAp side and can successfully prevent postoperative adhesion on the PLA side at the same time. Therefore, the HAp/PLA Janus membrane with its osteoinduction/barrier dual functions can be applied to repair bone defect precisely.

The inorganic part of human bone is mainly composed of hydroxyapatite (HAP: Ca10(PO4)6(OH)2) and whitlockite (WH: Ca18Mg2(HPO4)2(PO4)12) minerals, where the WH phase occupies up to 20–35% of total weight. These two bone minerals have different crystal structures and physicochemical properties, implying their distinguished role in bone physiology. However, until now, the biological significance of the presence of a certain ratio between HAP and WH in bone is unclear. To address this fundamental question, bone mimetic scaffolds are designed to encapsulate human mesenchymal stem cells (MSCs) for assessing their osteogenic activity depending on different ratios of HAP and WH. Interestingly, cellular growth and osteogenic differentiation are significantly promoted when MSCs are grown with a 3–1 ratio of HAP and WH nanoparticles, which is similar to bone. One of the reasons for this synergism between HAP and WH in hydrogel scaffolds is that, while WH nanoparticles can enhance osteogenic differentiation of MSCs compared to HAP, WH counterintuitively decreases the mechanical stiffness of nanocomposite hydrogels and hinders the osteogenic activity of cells. Taken together, these findings identify the optimal ratio between two major minerals in bone mimetic scaffolds to maximize the osteogenic differentiation of MSCs. Statement of significance Human bone minerals are composed of HAP and WH inorganic nanoparticles which have different material properties. However, the reason for the coexistence of HAP and WH in human bone is not fully identified, and HAP and WH composite biomaterial has not been utilized in the clinic. In this study, we have developed bone mimetic HAP and WH nanocomposite hydrogel scaffolds with various ratios. Importantly, we found out that HAP can promote the mechanical stiffness of the composite hydrogel scaffolds while WH can enhance the osteogenic activity of stem cells, which together induced synergism to maximize osteogenic differentiation of stem cells when mixed into 3–1 ratio that is similar to human bone.

Total joint replacements (TJR) are costly procedures required to relieve pain and restore function in patients suffering from end-stage arthritis. Despite great progress in the development and durability of TJRs, the generation of prosthesis-associated wear particles over time leads to an inflammatory cascade which culminates in periprosthetic osteolysis. Studies suggest that wear particles drive the polarization/differentiation of immature macrophages towards a pro-inflammatory M1 phenotype rather than an anti-inflammatory M2 phenotype associated with normal bone and wound healing. This, in turn, contributes to the initiation of peri-implant inflammation. As a result, modulating M1 macrophage cytokine production has been recognised as a viable therapeutic option. The aim of this study was to examine the impact of hydroxyapatite (HA) and poly(methyl methacrylate) (PMMA) particles on human macrophage polarization by comparing their effect on M1/M2-associated gene expression using real-time PCR. Furthermore, using immunoblotting to assess kinase activation, we sought to identify the intracellular signalling molecules activated by PMMA/HA particles and to determine whether pharmacological blockade of these molecules impacts on macrophage phenotype and cytokine production as measured by ELISA. We report that wear particles preferentially polarize macrophages towards an M1 phenotype, an effect that is dependent on activation of the membrane proximal kinase, Syk and members of the mitogen-activated protein kinase (MAPK) family of signalling molecules. Pre-treatment of macrophages with Syk inhibitors (R788/piceatannol) or MAPK inhibitors (SB203580 and PD98059), not only prevents M1 polarization, but also attenuates production of key pro-inflammatory mediators that have been specifically implicated in periprosthetic osteolysis and osteoclast differentiation. Statement of Significance It is now well established that wear-debris particles from implanted materials drive deleterious inflammatory responses which can eventually lead to implant loosening. In this study, we provide further insight into the specific cellular pathways activated by wear particles in primary human immune cells. We demonstrate that PMMA bone cement and hydroxyapatite, a commonly used biomaterial, drive the polarization of macrophages towards an inflammatory phenotype and identify the specific signalling molecules that are activated in this process. Pre-treatment of macrophages with pharmacological inhibitors of these molecules in turn prevents macrophage polarization and dampens inflammatory cytokine production. Hence these signalling molecules represent potential therapeutic targets to treat or possibly prevent particulate induced osteolysis.

Integration of in vitro -formed cartilage on a suitable substrate to form tissue-engineered implants for osteochondral defect repair is a considerable challenge. In healthy cartilage, a zone of calcified cartilage (ZCC) acts as an intermediary for mechanical force transfer from soft to hard tissue, as well as an effective interlocking structure to better resist interfacial shear forces. We have developed biphasic constructs that consist of scaffold-free cartilage tissue grown in vitro on, and interdigitated with, porous calcium polyphosphate (CPP) substrates. However, as CPP degrades, it releases inorganic polyphosphates (polyP) that can inhibit local mineralization, thereby preventing the formation of a ZCC at the interface. Thus, we hypothesize that coating CPP substrate with a layer of hydroxyapatite (HA) might prevent or limit this polyP release. To investigate this we tested both inorganic or organic sol-gel processing methods, as a barrier coating on CPP substrate to inhibit polyP release. Both types of coating supported the formation of ZCC in direct contact with the substrate, however the ZCC appeared more continuous in the tissue formed on the organic HA sol gel coated CPP. Tissues formed on coated substrates accumulated comparable quantities of extracellular matrix and mineral, but tissues formed on organic sol-gel (OSG)-coated substrates accumulated less polyP than tissues formed on inorganic sol-gel (ISG)-coated substrates. Constructs formed with OSG-coated CPP substrates had greater interfacial shear strength than those formed with ISG-coated and non-coated substrates. These results suggest that the OSG coating method can modify the location and distribution of ZCC and can be used to improve the mechanical integrity of tissue-engineered constructs formed on porous CPP substrates. Statement of Significance Articular cartilage interfaces with bone through a zone of calcified cartilage. This study describes a method to generate an “osteochondral-like” implant that mimics this organization using isolated deep zone cartilage cells and a sol-gel hydroxyapatite coated bone substitute material composed of calcium polyphosphate (CPP). Developing a layer of calcified cartilage at the interface should contribute to enhancing the success of this “osteochondral-like” construct following implantation to repair cartilage defects.

Adequate bone substitutes osseointegration has been difficult to achieve in osteoporosis. Hydroxyapatite of the osteoporotic bone, secreted by pathologic osteoblasts, had a smaller crystal size and lower crystallinity than that of the normal. To date, little is known regarding the interaction of synthetic hydroxyapatite nanoparticles (HANPs) with osteoblasts born in bone rarefaction. The present study investigated the biological effects of HANPs on osteoblastic cells derived from osteoporotic rat bone (OVX-OB), in comparison with the healthy ones (SHM-OB). A selective effect of different concentrations of HANPs on the two cell lines was observed that the osteoporotic osteoblasts had a higher tolerance. Reductions in cell proliferation, ALP activity, collagen secretion and osteoblastic gene expressions were found in the SHM-OB when administered with HANPs concentration higher than 25 µg/ml. In contrast, those of the OVX-OB suffered no depression but benefited from 25 to 250 µg/ml HANPs in a dose-dependent manner. We demonstrated that the different effects of HANPs on osteoblasts were associated with the intracellular calcium influx into the endoplasmic reticulum. The in vivo bone defect model further confirmed that, with a critical HANPs concentration administration, the osteoporotic rats had more and mechanically matured new bone formation than the non-treated ones, whilst the sham rats healed no better than the natural healing control. Collectively, the observed epigenetic regulation of osteoblastic cell function by HANPs has significant implication on defining design parameters for a potential therapeutic use of nanomaterials. Statement of Significance In this study, we investigated the biological effects of hydroxyapatite nanoparticles (HANPs) on osteoporotic rat bone and the derived osteoblast. Our findings revealed a previously unrecognized phenomenon that the osteoporotic individuals could benefit from higher concentrations of HANPs, as compared with the healthy individuals. The in vivo bone defect model confirmed that, with a critical HANPs concentration administration, the osteoporotic rats had more mechanically matured new bone formation than the non-treated ones, whilst the sham rats healed no better than the natural healing control. The selective effect of HANPs might be associated with the intracellular calcium influx into the endoplasmic reticulum. Collectively, the observed epigenetic regulation by HANPs has significant implication on defining design parameters for a potential therapeutic use of nanomaterials in a pathological condition.

In order to induce an efficient bone formation with human bone marrow mesenchymal stromal cells (hBMSC) associated to a scaffold, it is crucial to determine the key points of the hBMSC action after in vivo transplantation as well as the appropriate features of a scaffold. To this aim we compared the hBMSC behavior when grafted onto two biomaterials allowing different bone potential in vivo. The cancellous devitalized Tutoplast®-processed bone (TPB) and the synthetic hydroxyapatite/β-tricalcium-phosphate (HA/βTCP) which give at 6weeks 100% and 50% of bone formation respectively. We first showed that hBMSC adhesion is two times favored on TPB in vitro and in vivo compared to HA/βTCP. Biomaterial structure analysis indicated that the better cell adhesion on TPB is associated to its higher and smooth open pore architecture as well as its content in collagen. Our 6week time course analysis, showed using qPCR that only adherent cells are able to survive in vivo giving thus an advantage in term of cell number on TPB during the first 4weeks after graft. We then showed that grafted hBMSC survival is crucial as cells participate directly to bone formation and play a paracrine action via the secretion of hIGF1 and hRANKL which are known to regulate the bone formation and resorption pathways respectively. Altogether our results point out the importance of developing a smooth and open pore scaffold to optimize hBMSC adhesion and ensure cell survival in vivo as it is a prerequisite to potentiate their direct and paracrine functions.Around 10% of skeletal fractures do not heal correctly causing nonunion. An approach involving mesenchymal stromal cells (MSC) associated with biomaterials emerges as an innovative strategy for bone repair. The diversity of scaffolds is a source of heterogeneity for bone formation efficiency. In order to better determine the characteristics of a powerful scaffold it is crucial to understand their relationship with cells after graft. Our results highlight that a biomaterial architecture similar to cancellous bone is important to promote MSC adhesion and ensure cell survival in vivo. Additionally, we demonstrated that the grafted MSC play a direct role coupled to a paracrine effect to enhance bone formation and that both of those roles are governed by the used scaffold.

Controlling the phenotype of mesenchymal stem cells (MSCs) through the delivery of regulatory genes is a promising strategy in tissue engineering (TE). Essential to effective gene delivery is the choice of gene carrier. Non-viral delivery vectors have been extensively used in TE, however their intrinsic effects on MSC differentiation remain poorly understood. The objective of this study was to investigate the influence of three different classes of non-viral gene delivery vectors: (1) cationic polymers (polyethylenimine, PEI), (2) inorganic nanoparticles (nanohydroxyapatite, nHA) and (3) amphipathic peptides (RALA peptide) on modulating stem cell fate after reporter and therapeutic gene delivery. Despite facilitating similar reporter gene transfection efficiencies, these nanoparticle-based vectors had dramatically different effects on MSC viability, cytoskeletal morphology and differentiation. After reporter gene delivery (pGFP or pLUC), the nHA and RALA vectors supported an elongated MSC morphology, actin stress fibre formation and the development of mature focal adhesions, while cells appeared rounded and less tense following PEI transfection. These changes in MSC morphology correlated with enhanced osteogenesis following nHA and RALA transfection and adipogenesis following PEI transfection. When therapeutic genes encoding for transforming growth factor beta 3 (TGF-β3) and/or bone morphogenic protein 2 (BMP2) were delivered to MSCs, nHA promoted osteogenesis in 2D culture and the development of an endochondral phenotype in 3D culture, while RALA was less osteogenic and appeared to promote a more stable hyaline cartilage-like phenotype. In contrast, PEI failed to induce robust osteogenesis or chondrogenesis of MSCs, despite effective therapeutic protein production. Taken together, these results demonstrate that the differentiation of MSCs through the application of non-viral gene delivery strategies depends not only on the gene delivered, but also on the gene carrier itself. Statement of Significance Nanoparticle-based non-viral gene delivery vectors have been extensively used in regenerative medicine, however their intrinsic effects on mesenchymal stem cell (MSC) differentiation remain poorly understood. This paper demonstrates that different classes of commonly used non-viral vectors are not inert and they have a strong effect on cell morphology, stress fiber formation and gene transcription in MSCs, which in turn modulates their capacity to differentiate towards osteogenic, adipogenic and chondrogenic lineages. These results also point to the need for careful and tissue-specific selection of nanoparticle-based delivery vectors to prevent undesired phenotypic changes and off-target effects when delivering therapeutic genes to damaged or diseased tissues.

Critical limb ischemia impairs circulation to the extremities, causing pain, disrupted wound healing, and potential tissue necrosis. Therapeutic angiogenesis seeks to repair the damaged microvasculature directly to restore blood flow. In this study, we developed modular, micro-scale constructs designed to possess robust handling qualities, allow in vitro pre-culture, and promote microvasculature formation. The microbead matrix consisted of an agarose (AG) base to prevent aggregation, combined with cell-adhesive components of fibrinogen (FGN) and/or hydroxyapatite (HA). Microbeads encapsulating a co-culture of human umbilical vein endothelial cells (HUVEC) and fibroblasts were prepared and characterized. Microbeads were generally 80–100 µm in diameter, and the size increased with the addition of FGN and HA. Addition of HA increased the yield of microbeads, as well as the homogeneity of distribution of FGN within the matrix. Cell viability was high in all microbead types. When cell-seeded microbeads were embedded in fibrin hydrogels, HUVEC sprouting and inosculation between neighboring microbeads were observed over seven days. Pre-culture of microbeads for an additional seven days prior to embedding in fibrin resulted in significantly greater HUVEC network length in AG + HA + FGN microbeads, as compared to AG, AG + HA or AG + FGN microbeads. Importantly, composite microbeads resulted in more even and widespread endothelial network formation, relative to control microbeads consisting of pure fibrin. These results demonstrate that AG + HA + FGN microbeads support HUVEC sprouting both within and between adjacent microbeads, and can promote distributed vascularization of an external matrix. Such modular microtissues may have utility in treating ischemic tissue by rapidly re-establishing a microvascular network. Statement of Significance Critical limb ischemia (CLI) is a chronic disease that can lead to tissue necrosis, amputation, and death. Cell-based therapies are being explored to restore blood flow and prevent the complications of CLI. In this study, we developed small, non-aggregating agarose-hydroxyapatite-fibrinogen microbeads that contained endothelial cells and fibroblasts. Microbeads were easy to handle and culture, and endothelial sprouts formed within and between microbeads. Our data demonstrates that the composition of the microbead matrix altered the degree of endothelial sprouting, and that the addition of hydroxyapatite and fibrinogen resulted in more distributed sprouting compared to pure fibrin microbeads. The microbead format and control of the matrix formulation may therefore be useful in developing revascularization strategies for the treatment of ischemic disease.

Over the past few years, the development of novel adhesives for biological soft tissue adhesion has gained significant interest. Such adhesives should be non-toxic and biocompatible. In this study, we synthesized a novel solid adhesive using nanostructured hydroxyapatite (HAp) and evaluated its physical adhesion properties through in vitro testing with synthetic hydrogels and mouse soft tissues. The results revealed that HAp-nanoparticle dispersions and HAp-nanoparticle-assembled nanoporous plates showed efficient adhesion to hydrogels. Interestingly, the HAp plates showed different adhesive properties depending upon the shape of their nanoparticles. The HAp plate made up of 17nm-sized nanoparticles showed an adhesive strength 2.2times higher than that of the conventional fibrin glue for mouse skin tissues.The present study indicates a new application of inorganic biomaterials (bioceramics) as a soft tissue adhesive. Organic adhesives such as fibrin glues or cyanoacrylate derivatives have been commonly used clinically. However, their limited biocompatibility and/or low adhesion strength are some drawbacks that impair their clinical application. In this study, we synthesized a novel solid adhesive with biocompatible and biodegradable HAp nanoparticles without the aid of organic molecules, and showed a rapid and strong adhesion of mouse soft tissues compared to conventional fibrin glues. Given the importance of wet adhesion in biomedicine and biotechnology applications, our results will help not only in developing an efficient approach to close incised soft tissues, but also in finding novel ways to integrate soft tissues with synthetic hydrogels (such as drug reservoirs).

Fabrication of composite scaffolds using stereolithography (SLA) for bone tissue engineering has shown great promises. However, in order to trigger effective bone formation and implant integration, exogenous growth factors are commonly combined to scaffold materials. In this study, we fabricated biodegradable composite scaffolds using SLA and endowed them with osteopromotive properties in the absence of biologics. First we prepared photo-crosslinkable poly(trimethylene carbonate) (PTMC) resins containing 20 and 40 wt% of hydroxyapatite (HA) nanoparticles and fabricated scaffolds with controlled macro-architecture. Then, we conducted experiments to investigate how the incorporation of HA in photo-crosslinked PTMC matrices improved human bone marrow stem cells osteogenic differentiation in vitro and kinetic of bone healing in vivo. We observed that bone regeneration was significantly improved using composite scaffolds containing as low as 20 wt% of HA, along with difference in terms of osteogenesis and degree of implant osseointegration. Further investigations revealed that SLA process was responsible for the formation of a rich microscale layer of HA corralling scaffolds. To summarize, this work is of substantial importance as it shows how the fabrication of hierarchical biomaterials via surface-enrichment of functional HA nanoparticles in composite polymer stereolithographic structures could impact in vitro and in vivo osteogenesis. Statement of Significance This study reports for the first time the enhance osteopromotion of composite biomaterials, with controlled macro-architecture and microscale distribution of hydroxyapatite particles, manufactured by stereolithography. In this process, the hydroxyapatite particles are not only embedded into an erodible polymer matrix, as reported so far in the literature, but concentrated at the surface of the structures. This leads to robust in vivo bone formation at low concentration of hydroxyapatite. The reported 3D self-corralling composite architecture provides significant opportunities to develop functional biomaterials for bone repair and tissue engineering.

While the impact of substrate topographies at nano- and microscale on bone cell behavior has been particularly well documented, very few studies have analyzed the role of substrate closure at a tissular level. Moreover, these have focused on matrix deposition rather than on osteoblastic differentiation. In the present work, mouse calvaria cells were grown for 15 days on hydroxyapatite (HA) ceramics textured with three different macrogrooves shapes ( ** 100 µm): 1 sine and 2 triangle waveforms. We found that macrotopography favors cell attachment, and that bone-like tissue growth and organization are promoted by a tight “closure angle” of the substrate geometry. Interestingly, while Flat HA controls showed little marker expression at the end of the culture, cells grown on macrogrooves, and in particular the most closed (triangle waveform with a 517 µm spatial period) showed a fast time-course of osteoblast differentiation, reaching high levels of gene and protein expression of osteocalcin and sclerostin, a marker of osteocytes. Statement of Significance Many in vitro studies have been conducted on topography at nano and microscale, fewer have focused on the influence of macrotopography on osteoblasts. Ceramics with a controlled architecture were obtained throught a 3D printing process and used to assess osteoblast behavior. Biocompatible, they allowed the long-terme survival of osteoblast cells and the laying of an important bone matrix. V-shaped grooves were found to accelerates osteoblast differentiation and promote bone-like tissue deposition and maturation (osteocyte formation), proportionately to angle closure. Such macrostructures are attractive for the design of innovative implants for bone tissue engineering and in vitro models of osteogenesis.

The architectural features of synthetic bone grafts are key parameters for regulating cell functions and tissue formation for the successful repair of bone defects. In this regard, macroporous structures based on triply-periodic minimal surfaces (TPMS) are considered to have untapped potential. In the present study, custom-made implants based on a gyroid structure, with (GPRC) and without (GP) a cortical-like reinforcement, were specifically designed to fit an intended bone defect in rat femurs. Sintered hydroxyapatite implants were produced using a dedicated additive manufacturing technology and their morphological, physico-chemical and mechanical features were characterized. The implants' integrity and ability to support bone ingrowth were assessed after 4, 6 and 8 weeks of implantation in a 3-mm-long, femoral defect in Lewis rats. GP and GPRC implants were manufactured with comparable macro- to nano-architectures. Cortical-like reinforcement significantly improved implant effective stiffness and resistance to fracture after implantation. This cortical-like reinforcement also concentrated new bone formation in the core of the GPRC implants, without affecting newly formed bone quantity or maturity. This study showed, for the first time, that custom-made TPMS-based bioceramic implants could be produced and successfully implanted in load-bearing sites. Adding a cortical-like reinforcement (GPRC implants) was a relevant solution to improve implant mechanical resistance, and changed osteogenic mechanism compared to the GP implants. STATEMENT OF SIGNIFICANCE: Architectural features are known to be key parameters for successful bone repair using synthetic bioceramic bone graft. So far, conventional manufacturing techniques, lacking reproducibility and complete control of the implant macro-architecture, impeded the exploration of complex architectures, such as triply periodic minimal surfaces (TPMS), which are foreseen to have an unrivaled potential for bone repair. Using a new additive manufacturing process, macroporous TPMS-based bioceramics implants were produced in calcium phosphate, characterized and implanted in a femoral defect in rats. The results showed, for the first time, that such macroporous implants can be successfully implanted in anatomical load-bearing sites when a cortical-like outer shell is added. This outer shell also concentrated new bone formation in the implant center, without affecting new bone quantity or maturity.

Bioactive materials, able to induce hydroxyapatite precipitation in contact with body fluids, are of great interest for their bone bonding capacity. . The aim of this paper is to compare bioactive materials with different surface features to verify the mechanisms of action and the relationship with kinetics and type of precipitated hydroxyapatite over time. Four different surface treatments for Ti/Ti6Al4V alloy and a bioactive glass were selected and a different mechanism of bioactivity is supposed for each of them. Apart from the conventional techniques (FESEM, XPS and EDX), less common characterizations (zeta potential measurements on solid surfaces and FTIR chemical imaging) were applied. The results suggest that the OH groups on the surface have several effects: the total number of the OH groups mainly affects hydrophilicity of surfaces, while the isoelectric points, surface charge and ions attraction mainly depend on OH acidic/basic strength. Kinetics of hydroxyapatite precipitation is faster when it involves a mechanism of ion exchange while it is slower when it is due to electrostatic effects . The electrostatic effect cooperates with ion exchange and it speeds up kinetics of hydroxyapatite precipitation. Different bioactive surfaces are able to differently induce precipitation of type A and B of hydroxyapatite, as well as different degrees of crystallinity and carbonation. STATEMENT OF SIGNIFICANCE: The bone is made of a ceramic phase (a specific type of hydroxyapatite), a network of collagen fibers and the biological tissue. A strong bond of an orthopedic or dental implant with the bone is achieved by bioactive materials where precipitation and growth of hydroxyapatite occurs on the implant surface starting from the ions in the physiological fluids. Several bioactive materials are already known and used, but their mechanism of action is not completely known and the type of precipitated hydroxyapatite not fully investigated. In this work, bioactive titanium and bioglass surfaces are compared through conventional and innovative methodologies. Different mechanisms of bioactivity are identified, with different kinetics and the materials are able to induce precipitation of different types of hydroxyapatite, with different degree of crystallinity and carbonation.

In this study, we explored a ternary dopant system utilizing 0.25 wt% ZnO to induce osteogenesis, 0.5 wt% SiO