Zhidao Xia

A review on macrophage responses to biomaterials.

Macrophages are the dominant infiltrating cells that respond rapidly to biomaterial implantation in soft and hard tissues. These cells and their fused morphologic variants, multinucleated giant cells or foreign body giant cells, usually remain at biomaterial-tissue interfaces for the lifetime of the device in vivo. As a component of the immune system, macrophage activities are closely related to immune responses, inflammation and foreign body responses. However, macrophages also mediate biodegradation of bioresorbable materials via phagocytosis and extracellular degradation. In addition, macrophages are essential for effective tissue regeneration as they regulate the recruitment, proliferation and differentiation of target cells, such as fibroblasts, osteoblasts, endothelial cells and keratinocytes during healing processes.

Dose-dependent cytotoxicity of clinically relevant cobalt nanoparticles and ions on macrophages in vitro.

Despite the satisfactory short-term implant survivorship of metal-on-metal hip resurfacing arthroplasty, periprosthetic soft-tissue masses such as pseudotumours are being increasingly reported. Cytotoxic effects of cobalt or chromium have been suggested to play a role in its aetiology. The aim of this study was to investigate the effects of clinically relevant metal nanoparticles and ions on the viability of macrophages in vitro. A RAW 264.7 murine macrophage cell line was cultured in the presence of either: (1) cobalt, chromium and titanium nanoparticles sized 30-35 nm; or (2) cobalt sulphate and chromium chloride. Two methods were used to quantify cell viability: Alamar Blue assay and Live/Dead assay. The cytotoxicity was observed only with cobalt. Cobalt nanoparticles and ions demonstrated dose-dependent cytotoxic effects on macrophages in vitro: the cytotoxic concentrations of nanoparticles and ions were 1 x 10(12) particles ml(-1) and 1000 microM, respectively. The high concentration of cobalt nanoparticles required for cytotoxicity of macrophages in vitro suggests that increased production of cobalt nanoparticles in vivo, due to excessive MoM implant wear, may lead to local adverse biological effects. Therefore, cytotoxicity of high concentrations of metal nanoparticles phagocytosed by macrophages located in the periprosthetic tissues may be an important factor in pathogenesis of pseudotumours.

Differences between bisphosphonates in binding affinities for hydroxyapatite

Bisphosphonates (BPs) inhibit bone resorption and are widely used for the treatment of bone diseases, including osteoporosis. BPs are also being studied for their effects on hydroxyapatite (HAP)-containing biomaterials. There is a growing appreciation that there are hitherto unexpected differences among BPs in their mineral binding affinities that affect their pharmacological and biological properties. To study these differences, we have developed a method based on fast performance liquid chromatography using columns of HAP to which BPs and other phosphate-containing compounds can adsorb and be eluted by using phosphate buffer gradients at pH 6.8. The individual compounds emerge as discrete and reproducible peaks for a range of compounds with different affinities. For example, the peak retention times (min; mean +/- SEM) were 22.0 +/- 0.3 for zoledronate, 16.16 +/- 0.44 for risedronate, and 9.0 +/- 0.28 for its phosphonocarboxylate analog, NE10790. These results suggest that there are substantial differences among BPs in their binding to HAP. These differences may be exploited in the development of biomaterials and may also partly explain the extent of their relative skeletal retention and persistence of biological effects observed in both animal and clinical studies.

Characterization of metal-wear nanoparticles in pseudotumor following metal-on-metal hip resurfacing

UNLABELLED Biopsies from a typical case of pseudotumor following metal-on-metal hip resurfacing (MoMHR) were analyzed using light and transmission electron microscopy, backscatter scanning electron microscopy and energy dispersive x-ray spectrometry (EDS). Heavy macrophage infiltration was observed in all black pigmented specimens. Metal nanoparticles (NPs) were observed exclusively within phagosomes of living macrophages and fragments of dead macrophages. Although dead fibroblasts were found to be juxtaposed with dead and disintegrated macrophages, the NPs were not seen within either live or dead fibroblasts. Chromium (Cr) but not cobalt (Co) was the predominant component of the remaining wear NPs in tissue. The current study finding suggests that corrosion of Co in phagosomes of macrophages and resultant Co ion release lead to tissue necrosis and adverse soft tissue reactions (pseudotumors). Further studies are required to elucidate the precise mechanism of intracellular corrosion of metal NPs and the long-term toxicity of the Cr remaining in the peri-prosthetic tissues. FROM THE CLINICAL EDITOR In this study of metal-on-metal hip resurfacing-related tissue necrosis and pseudotumor formation, corrosion and decomposition of metallic cobalt in phagosomes of macrophages and resultant cobalt ion release were demonstrated to be the key elements of pathogenesis.

Proliferation and differentiation of human tenocytes in response to platelet rich plasma: An in vitro and in vivo study

Platelet rich plasma (PRP) is the autologous plasma fraction with a platelet‐rich cellular component which is enriched with a number of growth factors. Due to its availability and low cost, PRP has become an increasingly popular clinical tool as an alternative source of growth factors for various applications, for example, tendon regeneration but with limited success in clinical trials. The main objective of the current study was to determine whether activated PRP [i.e., platelet rich plasma‐clot release (PRCR)] could be used to induce the proliferation and collagen synthesis in human tenocyte in vitro. The advantage of using PRCR is that the platelet‐derived bioactive factors are more concentrated and could initiate a more rapid and accelerated healing response than PRP. Our results demonstrated that 10% PRCR treatment accelerated the extent of cell proliferation and collagen production by human tenocytes in vitro. The expression of specific tenocyte markers were similar to conventional fetal bovine serum (FBS)‐treated tenocytes implanted in mice within 14 days of implantation in diffusion chambers. Moreover, relatively more collagen fibrils were evident in PRCR‐treated tenocytes in vivo as compared to 10% FBS‐treated cells. Overall, our feasibility study has indicated that PRCR can induce human tenocyte proliferation and collagen synthesis which could be implemented for future tendon regeneration in reconstructive surgeries.

A novel nano-porous alumina biomaterial with potential for loading with bioactive materials.

Nano-porous alumina, with the potential for being loaded with bioactive materials, has been proposed as a novel material for coating implants. In this study, the shear strength of the interface between such nano-porous anodic aluminium oxide (AAO) coatings and titanium substrates, their biocompatibility, and their potential for pore loading have been investigated. An interface shear strength in excess of 29 MPa was obtained which is comparable with that of conventional plasma sprayed hydroxyapatite implant coatings. The viability and differentiation of MG63 osteoblastic cells co-cultured on the coating was found to be broadly comparable to that of similar cells co-cultured on conventional bioinert implant materials such as titanium and fully dense alumina. Extensive pore loading with silica nano-particles of different sizes and in different combinations was demonstrated throughout the thickness of AAO layers 1 microm and 60 microm thick. This work has demonstrated, that with suitable choice of pore filling materials, this novel coating might simultaneously combat infection, encourage bone regeneration, and secure fixation of the implant to bone.

Fluorescently Labeled Risedronate and Related Analogues: “Magic Linker” Synthesis

We report synthesis of the first fluorescently labeled conjugates of risedronate (1), using an epoxide linker strategy enabling conjugation of 1 via its pyridyl nitrogen with the label (carboxyfluorescein). Unlike prior approaches to create fluorescent bisphosphonate probes, the new linking chemistry did not abolish the ability to inhibit protein prenylation in vitro, while significantly retaining hydroxyapatite affinity. The utility of a fluorescent 1 conjugate in visualizing osteoclast resorption in vitro was demonstrated.

Stimulation of fibroblast growth in vitro by intermittent radiant warming.

A number of clinical studies have suggested that radiant heat improves the healing of selected acute and chronic wounds. The purpose of this study was to investigate in vitro the effect of intermittent radiant heating on the growth of human skin fibroblasts using a radiant heat‐producing dressing with a designated temperature of 38 °C. In initial experiments cells were seeded in six well‐plates, maintained in culture at 33–34 °C, and warmed daily for three cycles of 1 hour with 1.5 hour intervals. Changes in cell growth and metabolism were determined in sets of triplicate wells by cell counts and a colorimetric assay before and after one weeks treatment. After eight days the number of cells in the radiant heat‐treated group was 30% higher and the metabolic activity 47%– 90% higher than in the control group. In quiescent fibroblasts which had been maintained for four weeks in low‐serum medium, the warming regime completely prevented the decrease in cell number observed in control cells. Our findings suggest that the stimulation of cell proliferation induced by intermittent heating in vitro may indicate a possible mechanism contributing to in vivo effects.

Innate immune response to human bone marrow fibroblastic cell implantation in CB17 scid/beige mice.

Immunocompromised mouse models have been extensively used to assess human cell implantation for evaluation of cytotherapy, gene therapy and tissue engineering strategies, as these mice are deficient in T and B lymphoid cells. However, the innate immune response and its effect on human cell xenotransplantation in these mouse models are mainly unknown. The aim of this study is to characterise the myeloid populations in the spleen and blood of CB17 scid beige (CB17 sb) mice, and to study the inflammatory cell responses to xenogeneic implantation of enhanced green fluorescent protein (GFP)‐labelled human bone marrow fibroblastic (HBMF) cells into CB17 sb mice. The results indicate that even though CB17 sb mice are deficient in B‐ and T‐cells, they exhibit some increases in their monocyte (Mo), macrophage (MΦ) and neutrophil (Neu) populations. NK cell and eosinophil populations show no differences compared with wild‐type Balb/C mice. An innate immune response, identified by CR3 (CD11b/CD18)‐positive myeloid inflammatory cells and F4/80‐positive macrophages, was evident in the tissues where HBMF cells were implanted. As a consequence, the majority of implanted HBMF cells were eliminated by 4 weeks after implantation. Interestingly, the mineralised matrix formed by osteogenic HBMF cells was also eroded by multinuclear MΦ‐like giant cells. We conclude that CB17 sb mice retain active innate immune cells, which respond to HBMF cell xenotransplantation. This study highlights the importance of the innate immune cells in the anti‐xenograft response and suggests that strategies to block the activities of these cells may ameliorate the progressive long‐term elimination of xenotransplants. J. Cell. Biochem. 98: 966–980, 2006.

Adipogenic differentiation of adipose-derived stem cells in 3-dimensional spheroid cultures (microtissue): Implications for the reconstructive surgeon

BACKGROUND Adipose-derived stem cells (ADSC) can be readily extracted from adipose tissue, expanded in vitro, and have the capacity to differentiate into multiple cell lineages. This makes this cell type of great interest to the field of regenerative medicine. This study focuses on the isolation and characterisation of ADSC and their differentiation into adipocytes in a 3D microtissue model. METHODS Human ADSC were isolated from abdominal adipose tissue and characterised using multiparameter flow cytometry. ADSC were then expanded in culture and used to produce 3D scaffold-free micro-tissue. Adipogenic differentiation potential of micro-tissue constructs were subsequently characterised using Oil Red O staining. RESULTS Flow cytometric analysis showed ADSC were uniformly positive for CD34, CD73, CD90, and CD105, and negative for CD19, CD14, and CD45. The cells were functionally induced into adipocytes in the presence of appropriate conditioned media. CONCLUSION We have demonstrated that adipose-derived stem cells have the ability to form of microtissue and survive in vitro. We postulate that in the future this will result in an ADSC population which is injectable and can extend the delivery options of current stem cell-based therapies.