PLA/Hydroxyapatite scaffolds exhibit in vitro immunological inertness and promote robust osteogenic differentiation of human mesenchymal stem cells without osteogenic stimuli.

Bone defects stand out as one of the greatest challenges of reconstructive surgery. Fused deposition modelling (FDM) allows for the printing of 3D scaffolds tailored to the morphology and size of bone damage in a patient-specific and high-precision manner. However, FDM still suffers from the lack of materials capable of efficiently supporting osteogenesis. In this study, we developed 3D-printed porous scaffolds composed of polylactic acid/hydroxyapatite (PLA/HA) composites with high ceramic contents (above 20%, w/w) by FDM. The mechanical properties of the PLA/HA scaffolds were compatible with those of trabecular bone. In vitro degradation tests revealed that HA can neutralize the acidification effect caused by PLA degradation, while simultaneously releasing calcium and phosphate ions. Importantly, 3D-printed PLA/HA did not induce the upregulation of activation markers nor the expression of inflammatory cytokines in dendritic cells thus exhibiting no immune-stimulatory properties in vitro. Evaluations using human mesenchymal stem cells (MSC) showed that pure PLA scaffolds exerted an osteoconductive effect, whereas PLA/HA scaffolds efficiently induced osteogenic differentiation of MSC even in the absence of any classical osteogenic stimuli. Our findings indicate that 3D-printed PLA scaffolds loaded with high concentrations of HA are most suitable for future applications in bone tissue engineering.

Multifunctional hydroxyapatite and hopeite coatings on SS254 by laser rapid manufacturing for improved osseointegration and antibacterial character: A comparative study.

Orthopaedic metallic implant's long-term success strongly depends upon the two main factors: osseointegration and antibacterial character. Bioceramic (hydroxyapatite and hopeite) coatings have been proven effective for getting strong osseointegration and antibacterial character. However, deterioration of bioceramic coatings during the implantation period can adversely affect its overall biological performance. To conquer this issue, this research work recommends an innovative process route of laser rapid manufacturing for depositing bioceramic (hydroxyapatite and hopeite) coatings with metallurgical bonding. Microstructure, phase composition, antibacterial efficacy and bioactivity were evaluated using scanning electron microscopy, X-ray diffraction, fluorescence-activated cell sorting technique and simulated body fluid immersion test. The promising results obtained from these characterizations and testing establish the new process route laser rapid manufacturing as an effective alternative to deposit multifunctional bioceramic (hydroxyapatite and hopeite) coatings on metallic prosthetic-orthopaedic implants.

Rod-Scale Design Strategies for Immune-Targeted Delivery System toward Cancer Immunotherapy.

Strengthening the antitumor immune response to surpass the activation energy barrier associated with the immunosuppressive tumor microenvironment is an active area of cancer immunotherapy. Emerging evidence suggests that delivery of immunostimulatory molecules with the aid of a carrier system is essential for cancer immunotherapy. However, the size-dependent effect of the delivery system on immune-targeted sites and anticancer immune responses is yet to be comprehensively understood. Herein, to clarify the size-dependent effect of the delivery system on the underlying anticancer immune mechanism, rod-shaped hydroxyapatite (HA) particles with lengths from 100 nm to 10 µm are designed. HA rods stimulate anticancer immunity in a size-dependent manner. Shorter HA rods with lengths ranging from 100 to 500 nm promote antigen cellular uptake, dendritic cell (DC) maturation, and lymph node targeting antigen. In contrast, longer HA rods with lengths ranging from 500 nm to 10 µm prolong antigen retention and increase DC accumulation. Medium-sized HA rods with a length of 500 nm, taking advantage of both short and long rods, show optimized antigen release and uptake, increased DCs accumulation and maturation, highest CD4+ and CD8+ T cell population, and the best anticancer immunity in vivo. The present study provides a rod-scale design strategy for an immune-targeted delivery system toward cancer immunotherapy in the future.

Si-doping increases the adjuvant activity of hydroxyapatite nanorods.

Recombinant protein-based vaccines generally show limited immunogenicity and need adjuvants to achieve robust immune responses. Herein, to combine the excellent biocompatibility of hydroxyapatite (HA) and exciting adjuvant activity of silica, Si-doped HA nanorods with Si/P molar ratio from 0 to 0.65 were hydrothermally synthesized and evaluated as immunoadjuvants. Si-doping decreases the size and increases the BET surface area of the nanorods. Si-doping in HA nanorods increases the in vitro adjuvant activity, including CD11c+CD86+ expression and cytokine secretion of bone marrow derived dendritic cells (BMDCs). Moreover, Si-doping in HA increases the ex vivo adjuvant activity as shown by the increase in both Th1 and Th2 cytokines secretion. Si-doped HA nanorods are promising as a new immunoadjuvant.

In vivo immuno-reactivity analysis of the porous three-dimensional chitosan/SiO2 and chitosan/SiO2 /hydroxyapatite hybrids.

Inorganic/organic hybrid silica-chitosan (CS) scaffolds have promising potential for bone defect repair, due to the controllable mechanical properties, degradation behavior, and scaffold morphology. However, the precise in vivo immuno-reactivity of silica-CS hybrids with various compositions is still poorly defined. In this study, we fabricated the three-dimensional (3D) interconnected porous chitosan-silica (CS/SiO2 ) and chitosan-silica-hydroxyapatite (CS/SiO2 /HA) hybrids, through sol-gel process and 3D plotting skill, followed by the naturally or freeze drying separately. Scanning electron microscopy demonstrated the hybrids possessed the uniform geometric structure, while, transmission electron microscopy displayed nanoscale silica, or HA nanoparticles dispersed homogeneously in the CS matrix, or CS/silica hybrids. After intramuscular implantation, CS/SiO2 and CS/SiO2 /HA hybrids triggered a local and limited monocyte/macrophage infiltration and myofiber degeneration. Naturally dried CS/SiO2 hybrid provoked a more severe inflammation than the freeze-dried ones. Dendritic cells were attracted to invade into the implants embedded-muscle, but not be activated to prime the adaptive immunity, because the absence of cytotoxic T cells and B cells in muscle received the implants. Fluorescence-activated cell sorting (FACS) analysis indicated the implanted hybrids were incapable to initiate splenocytes activation. Plasma complement C3 enzyme linked immunosorbent assay (ELISA) assay showed the hybrids induced C3 levels increase in early implanting phase, and the subsequent striking decrease. Thus, the present results suggest that, in vivo, 3D plotted porous CS/SiO2 and CS/SiO2 /HA hybrids are relatively biocompatible in vivo, which initiate a localized inflammatory procedure, instead of a systematic immune response. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 1223-1235, 2018.

In vivo and in vitro evaluation of hydroxyapatite nanoparticle morphology on the acute inflammatory response.

Biomedical implants have been widely used in bone repair applications. However, nanosized degradation products from these implants could elicit an inflammatory reaction, which may lead to implant failure. It is well known that the size, chemistry, and charge of these nanoparticles can modulate this response, but little is known regarding the role that the particle's morphology plays in inducing inflammation. The present study aims to investigate the effect of hydroxyapatite nanoparticle (HANPs) morphology on inflammation, in-vitro and in-vivo. Four distinct HANP morphologies were fabricated and characterized: long rods, dots, sheets, and fibers. Primary human polymorphonuclear cells (PMNCs), mononuclear cells (MNCs), and human dermal fibroblasts (hDFs) were exposed to HANPs and alterations in cell viability, morphology, apoptotic activity, and reactive oxygen species (ROS) production were evaluated, in vitro. PMNCs and hDFs experienced a 2-fold decrease in viability following exposure to fibers, while MNC viability decreased 5-fold after treatment with the dots. Additionally, the fibers stimulated an elevated ROS response in both PMNCs and MNCs, and the largest apoptotic behavior for all cell types. Furthermore, exposure to fibers and dots resulted in greater capsule thickness when implanted subcutaneously in mice. Collectively, these results suggest that nanoparticle morphology can significantly impact the inflammatory response.

Nanobody-Based Electrochemical Immunoassay for Ultrasensitive Determination of Apolipoprotein-A1 Using Silver Nanoparticles Loaded Nanohydroxyapatite as Label.

Nanobodies (Nbs), derived from camelid heavy-chain antibodies, have distinct advantages over conventional antibodies in immunoassay. In this work, Nbs (Nb11 and Nb19) that can bind to different epitopes on apolipoprotein-A1 (Apo-A1) were screened out from an immunized Bactrian camel for the first time. Nb11 was used as capture antibody and fixed on gold nanoparticles (Au NPs) modified screen-printed carbon electrode (SPCE). The silver nanoparticles loaded nanohydroxyapatite (Ag-nHAP) was used as signal tag to label secondary antibody Nb19. A sandwich-type immunological reaction occurred between Apo-A1 and the two Nbs, which brought the Ag-nHAP to the SPCE surface. After the Ag-nHAP were acidically dissolved in the microelectrolytic cell of the SPCE, stripping voltammetric measurement for the released silver ions was performed to obtain an amplified signal. The peak current values increased by the logarithmic values of Apo-A1 concentrations from 10(-4) to 50 ng mL(-1) under optimal conditions. The detection limit was calculated to be 0.02 pg mL(-1). This method was used for the serum samples analysis and achieved satisfactory results. The low cost and high sensitivity make the electrochemical immunosensor suitable for the Apo-A1 detection, which may find promising application in other fields.

The soft tissue immunologic response to hydroxyapatite-coated transmucosal implant surfaces: a study in humans.

OBJECTIVE: To evaluate the soft tissue response in humans immunologically and histologically after placement of mini-implants coated with or without nano-size hydroxyapatite coatings. MATERIAL AND METHODS: Commercially pure (cp) titanium mini-implants (n = 13) or nano-hydroxyapatite-coated ones (n = 12) were randomly placed into partially edentulous jaws. Crevicular fluid was sampled 1 week after placement and subjected to quantitative polymerase chain reaction analysis to explore the inflammatory markers. After 8 weeks, implants and surrounding soft and hard tissue were trephined, and undecalcified ground sections were prepared. Inflammatory cell accumulation within a defined region of interest in the soft tissue was quantified histomorphometrically. RESULTS: No statistically significant differences in immunological response to the different implant surfaces were found for IL-6 (p = .438), TGF-ß2 (p = .467), MMP-8 (p = .758), CCL-3 (p = .758), IL-8 (p = .771), and IL-1ß (0.771). Histomorphometric evaluation presented no statistically significant difference between the two mini-implant surfaces with regards to number of inflammatory cells (p = .669). CONCLUSION: Nano-hydroxyapatite-coated surfaces in the transmucosal region yielded similar inflammatory response and is suggested to be as biocompatible as commercially pure titanium surfaces.

Randomized, placebo-controlled, double-blinded chemoimmunotherapy clinical trial in a pet dog model of diffuse large B-cell lymphoma.

PURPOSE: Active immunotherapy is a promising antitumoral strategy; however its use in combination with chemotherapy in dogs with large B-cell lymphoma (DLBCL) remains largely untested. Heat shock proteins (HSP) bind the small peptides they chaperone (HSPPC), allowing for immunization of the host against a large repertoire of tumor-associated antigens. Hydroxylapatite vehicles HSPPCs and acts as an immunologic adjuvant. The aim of this study was to show that an autologous vaccine with hydroxylapatite and tumor-derived HSPPCs is safe and therapeutically effective in dogs with DLBCL. EXPERIMENTAL DESIGN: Nineteen dogs with naturally occurring DLBCL were entered into a prospective randomized placebo-controlled double-blinded trial of HSPPCs-hydroxylapatite plus chemotherapy versus chemotherapy alone. Endpoints included time to progression (TTP), lymphoma-specific survival (LSS), and incidence of toxicoses. RESULTS: Median first TTP after randomization to the vaccine arm was 304 days versus 41 days for the control arm (P = 0.0004). There was also a statistically significant difference in duration of second remission between the two groups (P = 0.02). Median LSS was 505 days for the vaccinated dogs versus 159 days for the unvaccinated dogs (P = 0.0018). Six vaccinated dogs achieved molecular remission, as shown by clonal immunoglobulin H (IgH) rearrangement. Toxicoses were comparable between the two treatment arms. CONCLUSIONS: The results of this trial demonstrate that the autologous vaccine tested here is safe and efficacious in prolonging TTP and LSS in dogs with DLBCL when used in combination with dose-intense chemotherapy. On the basis of these results, additional evaluation of this novel therapeutic strategy is warranted in human DLBCL.

Calcium hydroxylapatite filler: an overview of safety and tolerability.

Soft tissue fillers are becoming increasingly important as nonsurgical treatment options for facial rejuvenation. Calcium hydroxylapatite (CaHA) is an injectable dermal filler that contains uniform CaHA microspheres suspended in an aqueous carboxymethylcellulose gel carrier. It is considered a long-lasting, but non-permanent filler, and is highly biocompatible with human tissue. No osteogenesis has been reported in extensive literature describing the use of CaHA in a variety of soft tissue applications. Injection of CaHA into the oral mucosa and the lips is an unapproved indication and may result in nodule formation. This occurs soon after injection and is a result of accumulated particles and not a granulomatous reaction. As with all biodegradable dermal fillers, CaHA can be associated with rare incidences of foreign body reactions, but only a handful of case reports have been documented in 10 years of clinical use. CaHA can be associated with local, short-term, injection-related adverse events, which are generally mild and resolve within a few days. Clinical trials that have followed patients for up to 3 years post-injection report no long-term or delayed-onset adverse events. CaHA is an effective and safe treatment option for a variety of aesthetic indications. This paper focuses on common safety concerns of patients and aesthetic physicians, including unfounded fears of osteogenesis and foreign body granulomas, providing an up-to-date overview of the tolerability and long-term safety of CaHA for aesthetic indications.

Cells-nano interactions and molecular toxicity after delayed hypersensitivity, in guinea pigs on exposure to hydroxyapatite nanoparticles.

The aim of the study was to evaluate the cells-nanoparticle interactions and molecular toxicity after delayed hypersensitivity in Guinea pigs, exposed to hydroxyapatite nanoparticles (HANP). The study focuses on synthesizing and characterizing HANPs and gaining an insight into the cytotoxicity, molecular toxicity, hypersensitivity and oxidative stress caused by them in vitro and in vivo. HANP was synthesized by chemical method and characterized by standard methods. Cytotoxicity was assessed on L929 cells by MTT assay and in vitro studies were carried out on rat liver homogenate. In vivo study was carried out by topical exposure of Guinea pigs with HANP, repeatedly, and evaluating the skin sensitization potential, blood parameters, oxidative stress in liver and brain and DNA damage (8-hydroxyl-2-deoxyguanosine: 8-OHdG) in liver. The results of the study indicated that there was no cytotoxicity (up to 600µg/mL) and oxidative damage (up to 100µg/mL), when exposed to HANPs. It was also evident that, there was no skin sensitization and oxidative damage when HANP were exposed to Guinea pigs.

Poly(vinyl alcohol)/collagen/hydroxyapatite hydrogel: properties and in vitro cellular response.

The objective of this study was to develop "bone-like" poly(vinyl alcohol) (PVA)/hydroxyapatite (HA)/type I collagen (Col) hydrogel composites that stimulate adhesion, proliferation, and differentiation of osteoblastic cells. The hydrogel composites were prepared by mixing PVA with nanoscale HA and Col using a physical mixing method. The concentration of the components was optimized during formulation development. PVA/Col/HA hydrogels were characterized for viscoelasticity, degree of swelling, mechanical strength, embedded erythromycin drug release, and cellular response of both osteoblastic MC3T3 cells and RAW 264.7 macrophage cells. Compressive strength tests confirmed that the PVA coating possessed greater elasticity and was mechanically enhanced by the freeze-thaw treatment. PVA/Col/HA gel is biocompatible and nontoxic to MC3T3 preosteoblasts, and the reinforcement from HA and Col reduced the inflammatory response from macrophages. Our findings demonstrate that PVA composites are biocompatible, and enhance cell adhesion, proliferation, and differentiation in vitro. We propose that PVA/Col/HA hydrogels represent one of the promising implant surface coating matrices for the improvement of implant osseointegration.

Conjugation of hydroxyapatite nanocrystals with human immunoglobulin G for nanomedical applications.

Inorganic nanosized drug carriers are a promising field in nanomedicine applied to cancer. Their conjugation with antibodies combines the properties of the nanoparticles themselves with the specific and selective recognition ability of the antibodies to antigens. Biomimetic carbonate-hydroxyapatite (HA) nanoparticles were synthesized and fully characterized; human IgGs, used as model antibodies, were coupled to these nanocrystals. The maximum loading amount, the interaction modelling, the preferential orientation and the secondary structure modifications were evaluated using theoretical models (Langmuir, Freundlich and Langmuir-Freundlich) spectroscopic (UV-Vis, Raman), calorimetric (TGA), and immunochemical techniques (ELISA, Western Blot). HA nanoparticles of about 30 nm adsorbed human IgGs, in a dose-dependent, saturable and stable manner with micromolar affinity and adsorption capability around 2.3 mg/m(2). Adsorption isotherm could be described by Langmuir-Freundlich model, and was due to both energetically homogeneous and heterogeneous binding sites on HA surface, mainly of electrostatic nature. Binding did not induce secondary structure modification of IgGs. A preferential IgG end-on orientation with the involvement of IgG Fc moiety in the adsorption seems most probable due to the steric hindrance of their Fab domains. Biomimetic HA nanocrystals are suitable substrates to produce nanoparticles which can be functionalized with antibodies for efficient targeted drug delivery to tumours.

Orthopedic implant of a polyhydroxybutyrate (PHB) and hydroxyapatite composite in cats.

The purpose of this study was to evaluate the tissue response to a 70% polyhydroxybutyrate and 30% hydroxyapatite composite in the form of a bone implant, placed intracortically in the distal metaphyseal of the right femur, and subcutaneous implants in cats. Samples of the composite were implanted subcutaneously in the dorsolumbar region and the distal metaphyseal region of the right femur of the animals. The study used 12 neutered adult mixed breed cats, weighing an average of 3.5kg. The cats were randomly divided into three groups: GI, GII and GIII, according to the length of the assessment period. The assessments of their subcutaneous and bone tissues were performed at 15, 30 and 45 days and at 30, 60 and 90 days, respectively. The subcutaneous and bone reactions to the composites were characterized by granulomatous inflammation with a predominance of macrophages and giant cells. The results showed that the composites triggered a chronic local inflammatory response, despite their clinical acceptance.

The effect of zinc on hydroxyapatite-mediated activation of human polymorphonuclear neutrophils and bone implant-associated acute inflammation.

Hydroxyapatite (HA) is widely used as coating biomaterial for prosthesis metal parts and as bone substitute. The release of HA particles induces an inflammatory response and, if uncontrolled, could result in implant loss. At the inflamed site, the polymorphonuclear cells (PMNs) represent the earliest phagocytic cells that predominate the cellular infiltrate. We have recently proposed that HA wear debris activate polymorphonuclear cells (PMNs) initiating and/or amplifying thereby the acute inflammatory response. Previous studies have shown that activation of monocytes by HA could be modulated by supplementing this latter with the divalent cation, Zinc. The purpose of this work was to investigate the modulation of PMNs activation following exposure to zinc-substituted HA. Our study demonstrate that addition of zinc to HA particles resulted in decreased levels of the pro-inflammatory mediator interleukin-8 (IL-8) and the matrix metallo-proteinase-9. We also show that these changes involve IL-8 receptors (CXCR-1 and CXCR-2).

Proinflammatory and osteoclastogenic effects of beta-tricalciumphosphate and hydroxyapatite particles on human mononuclear cells in vitro.

Particulate wear debris can activate defence cells and osteoclasts at the bone-implant interface possibly leading to bone resorption and implant failure. Cellular responses and inflammatory effects have been reported for particulate hydroxyapatite (HA). However, the immunological effects of particulate beta-tricalciumphosphate (beta-TCP) have not been studied and the question of whether beta-TCP is more biocompatible in this regard as is HA remains to be determined. Therefore the present work investigates effects of endotoxin-free HA and beta-TCP particles of the same size (d(50)=1 microm) and dose (SAR 10:1) on human peripheral blood mononuclear cells in vitro. The production of proinflammatory cytokines (TNF-alpha, IL-1beta, IL-8) and cytokines connected to osteoclast and dendritic cell differentiation (OPG, RANKL, M-CSF, GM-CSF) was determined by ELISA. After 6 and 18 h of incubation HA and beta-TCP caused a quite similar induction of TNF-alpha, IL-1beta and IL-8. Effects of particles on the production of M-CSF and OPG were not detectable. However, in sharp contrast to HA, beta-TCP caused less induction of GM-CSF and not any of RANKL, both known for promoting dendritic cells and osteoclastogenesis respectively. Therefore these in vitro data suggest that wear debris of beta-TCP poses lesser risk of the detrimental effects of osteoclast induction known from HA.

The biology of facial fillers.

The biologic behavior of a facial filler determines its advantages and disadvantages. The purpose of this article is to look at the relevant biology as part of a logical basis for making treatment decisions. Historical perspectives and biologic characteristics such as local tissue reaction (including phagocytosis and granulomatous inflammation) cross-linking, particle concentration, immunogenicity, biofilm formation, gel hardness, and collagen neogenesis are considered. Bovine collagen is the most immunogenic facial filler. Porcine and bioengineered human collagen implants have very low immunogenicity, but allergic reactions and elevations of IgG are possible. Cross-linking and concentration affect the longevity of collagen and hyaluronic acid fillers. Gel hardness affects how a hyaluronic acid filler flows through the syringe and needle. Calcium hydroxylapatite, poly-L-lactic acid, and polymethylmethacrylate fillers have been shown to stimulate collagen neogenesis. It appears that any facial filler can form a granuloma. Bacterial biofilms may play a role in the activation of quiescent granulomas. Various authors interpret the definition and significance of a granuloma differently.

Development of self-assembled nanoceramic carrier construct(s) for vaccine delivery.

Hydroxyapatite (HA) has been extensively investigated as scaffolds for tissue engineering, as drug delivery agents, as non-viral gene carriers, as prosthetic coatings, and composites. Recent studies in our laboratory demonstrated the immunoadjuvant properties of HA when administered with malarial merozoite surface protein-1(19) (MSP-1(19)). HA nanoceramic carrier was prepared by co-precipitation method that comprises of sintering and spray-drying technique. Prepared systems were characterized for crystallinity, size, shape, and antigen loading efficiency. Small size and large surface area of prepared HA demonstrated good adsorption efficiency of immunogens. Prepared nanoceramic formulations also showed slower in vitro antigen release and slower biodegrability behavior, which may lead to a prolonged exposure to antigen-presenting cells and lymphocytes. Furthermore, addition of mannose in nanoceramic formulation may additionally lead to increased stability and immunological reactions. Immunization with MSP-1(19) in nanoceramic-based adjuvant systems induced a vigorous immunoglobulin G (IgG) response, with higher IgG2a than IgG1 titers. In addition considerable amount of IFN-g and IL-2 was observed in spleen cells of mice immunized with nanoceramic-based vaccines. On the contrary, mice immunized with MSP-1(19) alone or with alum did not exhibit a significant cytotoxic response. The antibody responses to vaccine co-administered with HA was a mixed Th1/Th2 compared to the Th2-biased response obtained with alum. The prepared HA nanoparticles exhibit physicochemical properties that appear promising to make them a suitable immunoadjuvant to be used as antigen carriers for immunopotentiation.

In vivo analysis of biocompatibility and vascularization of the synthetic bone grafting substitute NanoBone.

One of the major challenges in the application of bone substitutes is adequate vascularization and biocompatibility of the implant. Thus, the temporal course of neovascularization and the microvascular inflammatory response of implants of NanoBone (fully synthetic nanocrystalline bone grafting material) were studied in vivo by using the mouse dorsal skinfold chamber model. Angiogenesis, microhemodynamics, and leukocyte-endothelial cell interaction were analyzed repetitively after implantation in the center and in the border zone of the implant up to 15 days. Both NanoBone granules and plates exhibited high biocompatibility comparable to that of cancellous bone, as indicated by a lack of venular leukocyte activation after implantation. In both synthetic NanoBone groups, signs of angiogenesis could be observed even at day 5 after implantation, whereas granules showed higher functional vessel density compared with NanoBone plates. The angiogenic response of the cancellous bone was markedly accelerated in the center of the implant tissue. Histologically, implant tissue showed an ingrowth of vascularized fibrous tissue into the material combined with an increased number of foreign-body giant cells. In conclusion, NanoBone, particularly in granular form, showed high biocompatibility and high angiogenic response, thus improving the healing of bone defects. Our results underline that, beside the composition and nanostructure, the macrostructure is also of importance for the incorporation of the biomaterial by the host tissue.

A reusable piezoelectric immunosensor using antibody-adsorbed magnetic nanocomposite.

This paper reports a simple, sensitive, and reusable piezoelectric immunosensor using magnetic hydroxyapatite (HAP)/gamma-Fe(2)O(3)/Au nanocomposite. Use of porous HAP nanocrystals embedded with gamma-Fe(2)O(3) and colloidal gold nanoparticles resulted in a multifunctional HAP/gamma-Fe(2)O(3)/Au nanocomposite. Under optimized conditions, the biocompatible nanocomposites were exploited for direct adsorption of large quantities of rabbit anti-human immunoglobulin G antibodies (anti-hIgG) with well-preserved immunoactivity. In a homogeneous bulk solution, the hIgG analytes were captured by the anti-hIgG-derivatized immunocomposites followed by magnetic separation/enrichment onto a bovine serum albumin (BSA)-sealed QCM probe before measuring. This QCM immunosensor can quantitatively determine concentrations of hIgG ranging from approximately 20 to 800 ng/ml, with a detection limit of approximately 15 ng/ml. Moreover, regeneration of the immunosensor can be simply realized by canceling the controllable magnetic field. With the possibility of performing the analysis automatically and considering its ease of use, high sensitivity, and good reusability, this magnetic separation-assisted QCM immunosensor may have great potential to be further tailored as a general and promising alternative for a broad range of practical applications.