Intramuscular Implantation Research Articles

Stimulatory effect of cobalt ions incorporated into calcium phosphate coatings on neovascularization in an in vivo intramuscular model in goats

Rapid vascularization of bone graft substitutes upon implantation is one of the most important challenges to overcome in order to achieve successful regeneration of large, critical-size bone defects. One strategy for stimulating vascularization during the regeneration process is to create a hypoxic microenvironment by either directly lowering the local oxygen tension, or by applying hypoxia-mimicking factors. Cells compensate for the hypoxic condition by releasing angiogenic factors leading to new blood vessel formation. In the present study, we explored the potential of cobalt ions (Co2+), known chemical mimickers of hypoxia, to stimulate vascularization within a bone graft substitute in vivo. To this end, Co2+ ions were incorporated into calcium phosphate (CaPs) coatings deposited on poly(lactic acid) (PLA) particles with their effect on the formation of new blood vessels studied upon intramuscular implantation in goats. PLA particles and CaP-coated particles without Co2+ ions served as controls. Pathological scoring of the inflammatory response following a 12-week implantation period showed no significant differences between the four types of materials. Based on histological and immunohistochemical analyses, both blood vessel area and number of blood vessels in CaP-coated PLA particles containing Co2+ were higher than in the uncoated PLA particles and CaP-coated PLA particles without Co2+. Analysis of blood vessel size distribution indicated abundant formation of small blood vessels in all the samples, while large blood vessels were predominantly found in PLA particles coated with CaP containing Co2+ ions. The results of this study support the use of CaPs containing Co2+ ions to enhance vascularization in vivo. Statement of SignificanceIn this work, we have investigated the potential of cobalt ions, incorporated into thin calcium phosphate (CaP) coatings that were deposited on particles of poly(lactic acid) (PLA), to induce neovascularization in vivo. Qualitative and quantitative histological and immunohistochemical analyses showed that both the number of blood vessels and the total blood vessel area were higher in CaP-coated PLA particles containing cobalt ions as compared to the uncoated PLA particles and CaP-coated PLA particles without the metallic additive. Furthermore, a wider distribution of blood vessel sizes, varying from very small to large vessels was specifically observed in samples containing cobalt ions.This in vivo study will significantly contribute to the existing knowledge on the use of bioinorganics, which are simple and inexpensive inorganic factors that can be used to control relevant biological process during tissue regeneration, such as vascularization. As such, we are convinced that this manuscript will be of interest to the readers of Acta Biomaterialia.

Mechanistic analysis of Zein nanoparticles/PLGA triblock in situ forming implants for glimepiride.

ObjectivesThe study aims at applying pharmaceutical nanotechnology and D-optimal fractional factorial design to screen and optimize the high-risk variables affecting the performance of a complex drug delivery system consisting of glimepiride–Zein nanoparticles and inclusion of the optimized formula with thermoresponsive triblock copolymers in in situ gel.MethodsSixteen nanoparticle formulations were prepared by liquid–liquid phase separation method according to the D-optimal fractional factorial design encompassing five variables at two levels. The responses investigated were glimepiride entrapment capacity (EC), particle size and size distribution, zeta potential, and in vitro drug release from the prepared nanoparticles. Furthermore, the feasibility of embedding the optimized Zein-based glimepiride nanoparticles within thermoresponsive triblock copolymers poly(lactide-co-glycolide)-block-poly(ethylene glycol)-block-poly(lactide-co-glycolide) in in situ gel was evaluated for controlling glimepiride release rate.ResultsThrough the systematic optimization phase, improvement of glimepiride EC of 33.6%, nanoparticle size of 120.9 nm with a skewness value of 0.2, zeta potential of 11.1 mV, and sustained release features of 3.3% and 17.3% drug released after 2 and 24 hours, respectively, were obtained. These desirability functions were obtained at Zein and glimepiride loadings of 50 and 75 mg, respectively, utilizing didodecyldimethylammonium bromide as a stabilizer at 0.1% and 90% ethanol as a common solvent. Moreover, incorporating this optimized formulation in triblock copolymers-based in situ gel demonstrated pseudoplastic behavior with reduction of drug release rate as the concentration of polymer increased.ConclusionThis approach to control the release of glimepiride using Zein nanoparticles/triblock copolymers-based in situ gel forming intramuscular implants could be useful for improving diabetes treatment effectiveness.

Biocompatibility and Biodegradation of Multiphasic Calcium Phosphate Ceramic Bone Substitute Transformed by Ostrich Cancellous Bone for Bone Tissue Engineering

A kind of novel multiphasic calcium phosphate ceramic bone substitute transformed by ostrich cancellous bone (OCB) was explored in this article. In this study we investigated the characterization, biocompatibility, biodegradability and in vivo osteoconductive property of the material. Scanning electron microscope (SEM) and X-ray diffraction (XRD) were used to characterize the material. Biocompatibility and biodegradation were confirmed by testing the hemolysis and intramuscular implantation. Bone marrow stromal cells (BMSCs) were also used to perform biocompatibility. Besides, material/BMSCs composites were implanted into the subcutaneous of back of nude mice to evaluate the osteoconductive property of the material. SEM analysis showed that the presence of a large amount of micropores within macropore walls and the interporous connections were abundant which indicated that the material possessed enough surface area. XRD pattern showed the peaks were corresponding to the characteristic for hydroxyapatite (HA), β-tricalcium phosphate (β-TCP), and sodium calcium phosphate (NaCaPO4). Culturing of BMSCs on the scaffolds revealed the cells were able to attach and proliferate easily on the surface and the pores of the scaffolds. Moreover, this scaffolds had a good performance of biodegradation in vivo. Finally, histological observation of heterotopic bone formation revealed newly formed bone at 8 weeks after transplantation. The results of this study demonstrated that the multiphasic calcium phosphate ceramic had good biocompatibility and its surface configuration and pore structure were suitable for BMSCs to attach and proliferate and also had a good osteoconductive property, which suggested that the material could be used as a bone tissue engineering scaffold in the future.

Using Biorelevant Testing to Characterize Release of Non Oral Dosage Forms as another Tool for Safety

Non-oral dosage forms often comprise of complex dosage forms such as parenterals, implants, drug eluting stents, transdermal patches, liposomes, microspheres and nano-particles as injectable. With the rapid advances in genetic engineering and recombinant DNA technology, the active drug moiety can also be a bio therapeutic (i.e. peptide or protein), which adds an additional layer of complexity to the drug product. In vitro dissolution tests/models for oral dosage forms such as tablets and suspensions have long been an indicator of in vivo performance of the drug. In commercial manufacturing processes, they act as an evaluative quality control tool. A dissolution test in oral dosage forms ensures minimal inter-batch variability and hence works as a check that the dosage form meets the requisite regulatory and quality standards. These in vitro models may also work as a tool for safety (i.e., predict in vivo performance and streamline clinical studies). The predictive in vitro data from these tests could often help in minimizing steps of clinical trials. In the case of non-oral dosage forms, similar tests that ensure performance verification of the drug are often called “in vitro release tests” rather than dissolution tests [1-3]. The rationale being non-oral dosage forms are frequently placed at diverse locations in the body e.g. transdermal patches on skin, drugeluting stents in coronary arteries [4], suppositories in rectum or urethra, subcutaneous or intramuscular implants (all of which have a different physiological environment). As a result, the action of the drug largely depends on its release from the dosage form. The drug moiety may be carried in complex delivery systems e.g. in a lipid based system such as liposomes, absorbable carrier scaffolds e.g. collagen matrix, injectable biodegradable polymer based matrix which solidifies at the site of action (e.g. ATRIGEL°). In cases like transdermal patches, the release needs to occur through multiple membranes and needs to penetrate through the skin and layers of external barriers before reaching the site of action. In subcutaneous and intramuscular implants the blood flow conditions might affect the time taken by the drug to reach the targeted site. Furthermore, the release mechanism, rate of release, and the time required by the drug to reach the site of action thus varies notably in each unique case. Hence the in vivo performance of these complex (non-oral) dosage forms can be characterized more precisely by carefully designed in vitro release tests, with design space criteria that are ‘biorelevant’. Unlike oral dosage forms where a single standardized USP method or apparatus can be used for dissolution testing of a class of compounds to determine in vivo performance (i.e., systemic release). Non-oral dosage forms can release locally with minimal systemic exposure. In such cases the standardized tests may not predict local release. The testing apparatus and method parameters have to be selected or modified accordingly to design a biorelevant reproducible and predictable in vitro release test. Biorelevance in Non-Oral Dosage Forms

Role of sodium potassium pump in osteoinduction process of calcium phosphate ceramic

Event Abstract Back to Event Role of sodium potassium pump in osteoinduction process of calcium phosphate ceramic Zhurong Tang1, Pengfei Xie1, Xiangdong Zhu1, Yujiang Fan1, Xingdong Zhang1 and Xiao Yang1 1 Sichuan University, National Engineering Research Center for Biomaterials, China Sodium potassium pump (Na+-K+ ATPase, NKA) is essential in regulation of intracellular calcium concentration. In a gene array study, Bone morphogenetic protein 2 (BMP2) was found to cause a 3-fold increment in gene expression of NKA subunit in human marrow stromal cells[1]. Thus, we hypothesized that the activation of NKA might be essential in bone forming process induced by calcium phosphate ceramics too. In vivo and in vitro experiments involving osteoinductive BCP bulk material (HA: beta-TCP = 2:8) with addition of several NKA inhibitor and activator agents were conducted. When co-cultured with BCP material, elevated gene expression of NKA alpha 1 and beta 4 subunits in mesenchymal stem cells (MSCs) were discovered. Furthermore, BMP2, OSX and BSP gene expressions in MSCs was significantly higher in the BCP material co-culture group than the cell alone control group. Adding in NKA specific inhibitors (ouabain or digoxin) in the BCP co-culture system will largely decrease the expression of BMP2 gene, whilst adding in NKA specific activator (SSA412) will increased BMP2 expression. Cell proliferation was not affected by any of these agents. Intramuscular implantation mouse model was used to evaluate ectopic bone formation induced by BCP alone and with exposure to NKA activator and inhibitors. Cylindrical BCP samples were immersed in ouabain, digoxin, SSA412 and vehicle solution before surgery. During the surgery, the BCP samples were implanted into the bilateral muscle pouches of each animal. The implants were retrieved after 3 month. The incidence of bone formation and bone area was significantly higher in SSA412 + BCP group. No pathological change was found in organs harvested. We conclude that the activity of NKA played an important in osteoinduction process. The authors wish to acknowledge the financial support from the National Science and Technology Supporting Project, Grant No. 2012BAI17B01; National Basic Research Program of China (973 project), Grant No. 2011CB606201.

Inflammatory and immuno-reactivity in mice induced by intramuscular implants of HSNGLPL peptide grafted-polyurethane.

Synthetic peptide-based polyurethanes (PUs), introduced as bioactive agents and possessing impressive properties, have emerged as attractive functional biomaterials for tissue regeneration. In this study, we developed a PU with a pendent HSNGLPL group through click reaction, which has strong affinity to TGF-β1. The peptide grafted-PUs, or PUs with BDO as the chain extender (control), were implanted into the gastrocnemius muscle (GN) of C57BL/6 mice, for evaluating their inflammatory and immuno-reactivity in vivo. We show herein that, after muscle implantation, BDO-PU induced a conspicuous monocyte/macrophage infiltration and myofiber degeneration. The inflammatory invasion and myofiber necrosis were mainly detected in the site around, but not far from, the implants, suggesting that the degraded PU matrix only triggers a local and limited inflammation in vivo. In contrast, peptide grafted-PU induced intramuscular inflammation was more complex and was sustained for more than 2 months. Apart from nonspecific monocyte/macrophage infiltration as in the case of BDO-PU, CD4+ T cells and dendritic cells (DCs), the members of the adaptive immune system, can be detected within the inflammatory site around peptide grafted-PU implants. The number of apoptotic macrophages in muscle containing peptide-PU was significantly lower compared to that in muscle containing BDO-PU. Thus, our present results suggest that, the PU matrix degradation-produced local environment is toxic to muscle cells and induces muscle degeneration. Moreover, highly aggregated peptide on PU might act as an immunogen to trigger intramuscular inflammation and lead to the delayed inflammatory response.

In vivo evaluation of the local inflammatory response after intramuscular implantation of Ti6Al4V plates with anti-adhesive plasma-fluorocarbon-polymer films in rats

In vivo evaluation of the local inflammatory response after intramuscular implantation of Ti6Al4V plates with anti-adhesive plasma-fluorocarbon-polymer films in rats

Safety of intradiscal injection and biocompatibility of polyester amide microspheres in a canine model predisposed to intervertebral disc degeneration.

Repair of degenerated intervertebral discs (IVD) might be established via intradiscal delivery of biologic therapies. Polyester amide polymers (PEA) were evaluated for in vitro cytotoxicity and in vivo biocompatibility, and thereafter intradiscal application of PEA microspheres (PEAMs) in a canine model predisposed to IVD degeneration at long‐term (6 months) follow‐up. PEA extracts did not induce cytotoxicity in mouse fibroblast cells (microscopy and XTT assay), while a slight foreign body reaction was demonstrated by histopathology after intramuscular implantation in rabbits. Intradiscal injection of a volume of 40 µL through 26 and 27G needles induced no degenerative changes in acanine model susceptible to IVD disease. Although sham‐injected IVDs showed increased CAV1 expression compared with noninjected IVDs, which may indicate increased cell senescence, these findings were not supported by immunohistochemistry, biomolecular analysis of genes related to apoptosis, biochemical and histopathological results. PEAM‐injected IVDs showed significantly higher BAX/BCL2 ratio vs sham‐injected IVDs suggestive of an anti‐apoptotic effect of the PEAMs. These findings were not supported by other analyses (clinical signs, disc height index, T2 values, biomolecular and biochemical analyses, and IVD histopathology). PEAs showed a good cytocompatibility and biocompatibility. PEAMs are considered safe sustained release systems for intradiscal delivery of biological treatments. © 2015 The Authors. Journal of Biomedical Materials Research Part B: Applied Biomaterials published by Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 707–714, 2017.

Poly(ethylene glycol) hydrogels with cell cleavable groups for autonomous cell delivery

Cell-responsive hydrogels hold tremendous potential as cell delivery devices in regenerative medicine. In this study, we developed a hydrogel-based cell delivery vehicle, in which the encapsulated cell cargo control its own release from the vehicle in a protease-independent manner. Specifically, we have synthesized a modified poly(ethylene glycol) (PEG) hydrogel that undergoes degradation responding to cell-secreted molecules by incorporating disulfide moieties onto the backbone of the hydrogel precursor. Our results show the disulfide-modified PEG hydrogels disintegrate seamlessly into solution in presence of cells without any external stimuli. The rate of hydrogel degradation, which ranges from hours to months, is found to be dependent upon the type of encapsulated cells, cell number, and fraction of disulfide moieties present in the hydrogel backbone. The differentiation potential of human mesenchymal stem cells released from the hydrogels is maintained in vitro. The in vivo analysis of these cell-laden hydrogels, through a dorsal window chamber and intramuscular implantation, demonstrated autonomous release of cells to the host environment. The hydrogel-mediated implantation of cells resulted in higher cell retention within the host tissue when compared to that without a biomaterial support. Biomaterials that function as a shield to protect cell cargos and assist their delivery in response to signals from the encapsulated cells could have a wide utility in cell transplantation and could improve the therapeutic outcomes of cell-based therapies.

Surface heparin treatment of the decellularized porcine heart valve: Effect on tissue calcification.

Tissue calcification is a major cause of failure of bioprosthetic heart valves. Aim of this study was to examine whether surface heparin treatment of the decellularized porcine heart valve reduces tissue calcification. Fresh porcine aortic heart valves were dissected as tissue discs and divided into four groups. Group A: controls without treatment, Group B: decellularization only, Group C: decellularization and glutaraldehyde cross-linking, Group D: decellularization and glutaraldehyde cross-linking followed by surface heparin treatment. After implantation in New Zealand White rabbits for 60 days, the explanted heart valve discs from the different study groups underwent a series of histological examinations as well as determination of calcium content by the methyl thyme phenol blue colorimetric method. Results of the explanted heart valve discs for the Von Kossa staining demonstrated that in Group A the heart valve tissue was the most severely stained with black color, whereas in Group D there was hardly any area that was stained black after implantation indicating the least tissue calcification. Furthermore, the inflammatory cells identified by the Hematoxylin-eosin staining appeared to be the least in Group D. The average tissue calcium content was highest in Group A (0.197 ± 0.115 μmolmg-1 ), modest in Group B (0.113 ± 0.041 μmolmg-1 ), and Group C (0.089 ± 0.049 μmolmg-1 ), and the lowest in Group D (0.019 ± 0.019 μmolmg-1 , p < 0.05). These results suggest that surface heparin treatment tends to reduce tissue calcification of the dellellularized porcine heart valve in a rabbit intramuscular implantation model. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 400-405, 2017.

In vivo demonstration of injectable microstimulators based on charge-balanced rectification of epidermically applied currents

Objective. It is possible to develop implantable microstimulators whose actuation principle is based on rectification of high-frequency (HF) current bursts supplied through skin electrodes. This has been demonstrated previously by means of devices consisting of a single diode. However, previous single diode devices caused dc currents which made them impractical for clinical applications. Here flexible thread-like stimulation implants which perform charge balance are demonstrated in vivo. Approach. The implants weigh 40.5 mg and they consist of a 3 cm long tubular silicone body with a diameter of 1 mm, two electrodes at opposite ends, and, within the central section of the body, an electronic circuit made up of a diode, two capacitors, and a resistor. In the present study, each implant was percutaneously introduced through a 14 G catheter into either the gastrocnemius muscle or the cranial tibial muscle of a rabbit hindlimb. Then stimulation was performed by delivering HF bursts (amplitude <60 V, frequency 1 MHz, burst repetition frequency from 10 Hz to 200 Hz, duration = 200 μs) through a pair of textile electrodes strapped around the hindlimb and either isometric plantarflexion or dorsiflexion forces were recorded. Stimulation was also assayed 1, 2 and 4 weeks after implantation. Main results. The implants produced bursts of rectified current whose mean value was of a few mA and were capable of causing local neuromuscular stimulation. The implants were well-tolerated during the 4 weeks. Significance. Existing power supply methods, and, in particular inductive links, comprise stiff and bulky parts. This hinders the development of minimally invasive implantable devices for neuroprostheses based on electrical stimulation. The proposed methodology is intended to relieving such bottleneck. In terms of mass, thinness, and flexibility, the demonstrated implants appear to be unprecedented among the intramuscular stimulation implants ever assayed in vertebrates.

BMP-2 Derived Peptide and Dexamethasone Incorporated Mesoporous Silica Nanoparticles for Enhanced Osteogenic Differentiation of Bone Mesenchymal Stem Cells.

Bone morphogenetic protein-2 (BMP-2), a growth factor that induces osteoblast differentiation and promotes bone regeneration, has been extensively investigated in bone tissue engineering. The peptides of bioactive domains, corresponding to residues 73-92 of BMP-2 become an alternative to reduce adverse side effects caused by the use of high doses of BMP-2 protein. In this study, BMP-2 peptide functionalized mesoporous silica nanoparticles (MSNs-pep) were synthesized by covalently grafting BMP-2 peptide on the surface of nanoparticles via an aminosilane linker, and dexamethasone (DEX) was then loaded into the channel of MSNs to construct nanoparticulate osteogenic delivery systems (DEX@MSNs-pep). The in vitro cell viability of MSNs-pep was tested with bone mesenchymal stem cells (BMSCs) exposure to different particle concentrations, revealing that the functionalized MSNs had better cytocompatibility than their bare counterparts, and the cellular uptake efficiency of MSNs-pep was remarkably larger than that of bare MSNs. The in vitro results also show that the MSNs-pep promoted osteogenic differentiation of BMSCs in terms of the levels of alkaline phosphatase (ALP) activity, calcium deposition, and expression of bone-related protein. Moreover, the osteogenic differentiation of BMSCs can be further enhanced by incorporating of DEX into MSNs-pep. After intramuscular implantation in rats for 3 weeks, the computed tomography (CT) images and histological examination indicate that this nanoparticulate osteogenic delivery system induces effective osteoblast differentiation and bone regeneration in vivo. Collectively, the BMP-2 peptide and DEX incorporated MSNs can act synergistically to enhance osteogenic differentiation of BMSCs, which have potential applications in bone tissue engineering.

Influence of surface microstructure and chemistry on osteoinduction and osteoclastogenesis by biphasic calcium phosphate discs.

It has been reported that surface microstructural dimensions can influence the osteoinductivity of calcium phosphates (CaPs), and osteoclasts may play a role in this process. We hypothesised that surface structural dimensions of ≤ 1 μm trigger osteoinduction and osteoclast formation irrespective of macrostructure (e.g., concavities, interconnected macropores, interparticle space) or surface chemistry. To test this, planar discs made of biphasic calcium phosphate (BCP: 80% hydroxyapatite, 20% tricalcium phosphate) were prepared with different surface structural dimensions - either ~ 1 μm (BCP1150) or ~ 2-4 μm (BCP1300) - and no macropores or concavities. A third material was made by sputter coating BCP1150 with titanium (BCP1150Ti), thereby changing its surface chemistry but preserving its surface structure and chemical reactivity. After intramuscular implantation in 5 dogs for 12 weeks, BCP1150 formed ectopic bone in 4 out of 5 samples, BCP1150Ti formed ectopic bone in 3 out of 5 samples, and BCP1300 formed no ectopic bone in any of the 5 samples. In vivo, large multinucleated osteoclast-like cells densely colonised BCP1150, smaller osteoclast-like cells formed on BCP1150Ti, and osteoclast-like cells scarcely formed on BCP1300. In vitro, RAW264.7 cells cultured on the surface of BCP1150 and BCP1150Ti in the presence of osteoclast differentiation factor RANKL (receptor activator for NF-κB ligand) proliferated then differentiated into multinucleated osteoclast-like cells with positive tartrate resistant acid phosphatase (TRAP) activity. However, cell proliferation, fusion, and TRAP activity were all significantly inhibited on BCP1300. These results indicate that of the material parameters tested - namely, surface microstructure, macrostructure, and surface chemistry - microstructural dimensions are critical in promoting osteoclastogenesis and triggering ectopic bone formation.

Development and Characterization of SMEDDS Based Thermally Triggered In Situ Gelling Intramuscular Implant for Sustained Release of Rifampicin

Development and Characterization of SMEDDS Based Thermally Triggered In Situ Gelling Intramuscular Implant for Sustained Release of Rifampicin

Flexible multi-channel microelectrode with fluidic paths for intramuscular stimulation and recording

Biomedical microelectrodes play a significant role in motor paralysis recovery caused by spinal cord injury (SCI). As most researchers focused on implantable microelectrodes with multiple functions for neural studies and application, we developed flexible intramuscular microelectrodes integrated wire electrodes for functional electrical stimulation (FES) and electromyogram (EMG) recording with micro tube channels for fluidic drug delivery. The electrodes were electrochemically deposited with conducting polymer (PEDOT/pTS) coating to improve the electrode performance including electrochemical impedance and charge storage capacity (CSC). Moreover, the excellent mechanical and electrochemical stability of PEDOT/pTS coated electrodes was verified by withstanding ultrasonic bath and experiencing repeated cyclic voltammetry (CV) scan. Furthermore, the fluidic property of the micro channels of electrodes was investigated to be suitable for intramuscular drug delivery. In addition, electrophysiological experiments in vivo comprising FES and EMG recording were performed to evaluate the practical application of flexible intramuscular microelectrodes. As a consequence, the developed multi-functional intramuscular microelectrodes are suitable for intramuscular implantation and electrophysiological application, and open a new gate for future researchers on intramuscular motor prostheses for paralysis recovery.

Systemic IFNγ predicts local implant macrophage response.

Implantation of biomaterials can cause complications often associated with inflammatory reactions. However, repeated evaluation of the implant site would be burdening for patients. Alternatively, blood examinations with analysis of inflammatory serum markers could potentially be useful to reflect the local cellular response for detection and/or prediction of inflammation-related complications. Therefore, following intramuscular implantation of surface-modified Ti implants in rats, this study aimed at examining possible associations between the post-implantation time course of pro-inflammatory (INFγ, IL-2) and anti-inflammatory (IL-4, IL-10) cytokine serum concentrations and the local peri-implant tissue response after 56 days (pro-inflammatory CD68-positive monocytes/macrophages, anti-inflammatory CD163-positive macrophages, MHC class II-positive cells, activated natural killer cells and mast cells). Multivariate correlation analysis revealed a significant interaction between serum IFNγ and peri-implant tissue CD68-positive monocytes/macrophages (p = 0.001) while no interactions were found for other cytokines and cell types. Additional Pearson correlation analysis of IFNγ serum concentrations on each experimental day vs. the CD68-positive monocytes/macrophages response on day 56 demonstrated a consistently positive correlation that was strongest during the first three weeks. Thus, high early pro-inflammatory IFNγ serum concentration was associated with high late number of pro-inflammatory CD68-positive monocyte/macrophages and low early serum IFNγ with low late CD68-positive monocyte/macrophage numbers. Further studies aimed at examination of patient samples could establish the relevance of this association to predict clinical complications. After implantation of titanium samples, high early IFNγ serum concentrations were associated with a pronounced late pro-inflammatory CD68-positive monocyte/ macrophage (red circle) response, while no correlation was found for other investigated cytokines and inflammatory cells (green circle). In contrast, low early IFNγ serum concentrations were correlated with low late monocyte/ macrophage numbers.

Effect of crystallinity and plasticizer on mechanical properties and tissue integration of starch-based materials from two botanical origins

The application of starch-based materials for biomedical purposes has attracted significant interest due to their biocompatibility. The physical properties and crystal structure of materials based on potato starch (PS) and amylomaize starch (AMS) were studied under physiological conditions. PS plasticized with 20% glycerol presented the best mechanical properties with an elastic modulus of 1.6MPa and a weak swelling, remaining stable for 30 days. The in vitro cell viability of 3T3 cells after contact with extracts from PS and AMS with 20% glycerol is 72% and 80%, respectively. PS presented good tissue integration and no significant inflammation or foreign body response after 30 days intra-muscular implantation in a rat model, contrary to AMS. It was shown that glycerol plasticization favors a fast B-type crystallization of PS materials, enhancing their mechanical strength and durability, and making them a good candidate for bioresorbable and biocompatible materials for implantable medical devices.

Biocompatibility of Bletilla striata Microspheres as a Novel Embolic Agent.

We have prepared Chinese traditional herb Bletilla striata into microspheres as a novel embolic agent for decades. The aim of this study was to evaluate the biocompatibility of Bletilla striata microspheres (BSMs). After a thermal test of BSMs in vitro, the cell biocompatibility of BSMs was investigated in mouse fibroblasts and human umbilical vein endothelial cells using the methyl tetrazolium (MTT) assay. In addition, blood biocompatibility was evaluated. In vivo intramuscular implantation and renal artery embolization in rabbits with BSMs were used to examine the inflammatory response. The experimental rabbits did not develop any fever symptoms after injection of BSMs, and BSMs exhibited no cytotoxicity in cultured mouse fibroblasts and human umbilical vein endothelial cells. Additionally, BSMs exhibited high compatibility with red blood cells and no hemolysis activity. Intramuscular implantation with BSMs resulted in a gradually lessened mild inflammatory reaction that disappeared after eight weeks. The occlusion of small renal vessels was associated with a mild perivascular inflammatory reaction without significant renal and liver function damage. In conclusion, we believe that BSMs exhibit high biocompatibility and are a promising embolic agent.

Local intramuscular transplantation of autologous mononuclear cells for critical lower limb ischaemia.

Peripheral arterial disease is a major health problem, and in about 1% to 2% of patients the disease progresses to critical limb ischaemia (CLI). In a substantial number of patients with CLI, no effective treatment option other than amputation is available and around a quarter of these patients will require a major amputation during the following year. This is an update of the review first published in 2011. To determine the effectiveness and safety of local intramuscular transplantation of autologous adult bone marrow mononuclear cells (BMMNCs) as a treatment for critical limb ischaemia (CLI). For this update the Cochrane Peripheral Vascular Diseases Group Trials Search Co-ordinator searched the Specialised Register (last searched February 2014) and the Cochrane Central Register of Controlled Trials (CENTRAL; 2014, Issue 1). We included all randomised controlled trials of CLI in which participants were randomly allocated to intramuscular administration of autologous adult BMMNCs or control (either no intervention or conventional conservative therapy). We excluded studies on patients with intermittent claudication. Two authors independently selected trials, assessed trials for eligibility and methodological quality, and extracted data. Disagreements were resolved by consensus or by the third author. Only two small studies, with a combined total of 57 participants, met our inclusion criteria and were finally included. They were classified as having a moderate risk of bias with unclear issues regarding their methods, and according to the GRADE approach, the overall quality of the evidence would be considered as moderate. In one study the effects of intramuscular injections of BMMNCs in the ischaemic lower limbs of patients with CLI were compared with control (standard conservative treatment). No deaths were reported and no significant difference was observed between the two groups for either pain (P = 0.37) or the ankle brachial index (ABI) parameter. However, the treatment group showed a significantly smaller proportion of participants undergoing amputation compared with the control group (P = 0.026).In the other study, following subcutaneous injections of granulocyte colony-stimulating factor (G-CSF) for five days, peripheral blood derived mononuclear cells were collected and then transplanted by intramuscular injections into ischaemic lower limbs. The effects were compared with daily intravenous prostaglandin E1 injections (control group). No deaths were reported. Pain reduction was greater in the treatment group than in the control group (P < 0.001) as was increase in ABI (mean increase 0.13 versus 0.02, P < 0.01). The treatment group experienced a statistically significant increase in pain-free walking distance (PFWD) compared with the control group (mean increase 306.4 m versus 78.6 m, P = 0.007). A smaller proportion of participants underwent amputation in the treatment group compared with the control group (0% versus 36%, P = 0.007). The data from the published trials suggest that there is insufficient evidence to support this treatment. These results were based on only two trials which had a very small number of participants. Therefore evidence from larger randomised controlled trials is needed in order to provide adequate statistical power to assess the role of intramuscular mononuclear cell implantation in patients with CLI.

Toxicity assessment of a novel silk fibroin and poly-methyl-methacrylate composite material

Based on its biocompatibility, silk fibroin has been applied in the fabrication of biomaterials in various forms such as gels, powders, fibers, and membranes. Poly-methyl-methacrylate (PMMA), which has been successfully used as a filler material in vertebroplasty, has the disadvantage of excessive inherent stiffness, which may damage adjacent vertebral bodies in elderly patients with osteoporosis. A novel potential filler material, consisting of silk fibroin and PMMA (silk/PMMA), was tested to assess its cytotoxicity, genotoxicity, and intramuscular implantation experiment according to ISO10993. The MTT assay, Ames test, mouse lymphoma assay, and intramuscular implantation were performed using extracts from solidified silk/PMMA, according to the protocol in ISO10993. Extracts of silk/PMMA did not show cytotoxicity, genotoxicity or toxic reaction in intramuscular implantation. These findings suggest that the silk/PMMA composite materials may potentially be used as a non-toxic biomaterial.