Apicomplexan parasites are attenuated by low-energy electron irradiation in an automated microfluidic system and protect against infection with Toxoplasma gondii.

Radiation-attenuated intracellular parasites are promising immunization strategies. The irradiated parasites are able to invade host cells but fail to fully replicate, which allows for the generation of an efficient immune response. Available radiation technologies such as gamma rays require complex shielding constructions and are difficult to be integrated into pharmaceutical production processes. In this study, we evaluated for the first time low-energy electron irradiation (LEEI) as a method to generate replication-deficient Toxoplasma gondii and Cryptosporidium parvum. Similar to other radiation technologies, LEEI mainly damages nucleic acids; however, it is applicable in standard laboratories. By using a novel, continuous, and microfluidic-based LEEI process, tachyzoites of T. gondii and oocysts of C. parvum were irradiated and subsequently analyzed in vitro. The LEEI-treated parasites invaded host cells but were arrested in intracellular replication. Antibody-based analysis of surface proteins revealed no significant structural damage due to LEEI. Similarly, excystation rates of sporozoites from irradiated C. parvum oocysts were similar to those from untreated controls. Upon immunization of mice, LEEI-attenuated T. gondii tachyzoites induced high levels of antibodies and protected the animals from acute infection. These results suggest that LEEI is a useful technology for the generation of attenuated Apicomplexan parasites and has potential for the development of anti-parasitic vaccines.

Sterilization and Cross-Linking Combined with Ultraviolet Irradiation and Low-Energy Electron Irradiation Procedure: New Perspectives for Bovine Pericardial Implants in Cardiac Surgery.

BACKGROUND: Bovine pericardium is the major natural source of patches and aortic valve substitutes in cardiac repair procedures. However, long-term tissue durability and biocompatibility issues lead to degeneration (e.g., calcification) that requires reoperation. Tissue preparation strategies, including glutaraldehyde fixation, are reasons for the deterioration of pericardial tissues. We describe a pretreatment procedure involving sterilization and cross-linking combined with ultraviolet (UV) irradiation and low-energy electron irradiation (SULEEI). This innovative, glutaraldehyde-free protocol improves the mechanical aspects and biocompatibility of porcine pericardium patches. METHODS: We adopted the SULEEI protocol, which combines decellularization, sterilization, and cross-linking, along with UV irradiation and low-energy electron irradiation, to pretreat bovine pericardium. Biomechanics, such as ultimate tensile strength and elasticity, were investigated by comparing SULEEI-treated tissue with glutaraldehyde-fixed analogues, clinical patch materials, and an aortic valve substitute. Histomorphological and cellular aspects were investigated by histology, DNA content analysis, and degradability. RESULTS: Mechanical parameters, including ultimate tensile strength, elasticity (Young's modulus), and suture retention strength, were similar for SULEEI-treated and clinically applied bovine pericardium. The SULEEI-treated tissues showed well-preserved histoarchitecture that resembled all pericardial tissues investigated. Fiber density did not differ significantly. DNA content after the SULEEI procedure was reduced to less than 10% of the original tissue material, and more than 50% of the SULEEI-treated pericardium was digested by collagenase. CONCLUSION: The SULEEI procedure represents a new treatment protocol for the preparation of patches and aortic valve prostheses from bovine pericardial tissue. The avoidance of glutaraldehyde fixation may lessen the tissue degeneration processes in cardiac repair patches and valve prostheses.

Growth Factor-Bearing Polymer Brushes--Versatile Bioactive Substrates Influencing Cell Response.

In this study we present the development of responsive nanoscale substrates exhibiting cell-guiding properties based on incorporated bioactive signaling cues. The investigative approach considered the effect of two different surface-bound growth factors (GFs) on cell behavior and response: hepatocyte growth factor (HGF) and basic fibroblast growth factor (bFGF). Two surface biofunctionalization strategies were explored in order to conceive versatile, bioactive thin polymer brush films. Polymer brushes made of tethered poly(acrylic)acid (PAA) polymer layers with a high grafting density of polymer chains were biofunctionalized with GFs either by physisorption or chemisorption. Both GFs showed high binding efficiencies to PAA brushes based on their initial loading concentrations. The GF release kinetics can be distinguished depending on the applied biofunctionalization method. Specifically, a high initial burst followed by a constant slow release was observed in the case of both physisorbed HGF and bFGF. In contrast, the release kinetics of chemisorbed GFs were quite different. Remarkably, chemisorbed HGF remained bound to the brush surface for over 1 week, whereas 50% of chemisorbed bFGF was released slowly. Furthermore, the effect of these GF-biofunctionalized PAA brushes on different cells was investigated. A human hepatoma cell line (HepG2) was used to analyze the bioactivity of HGF-modified PAA brushes by measuring cell growth inhibition and scattering effects. Additionally, the differentiation of mouse embryonic stem cells (mESCs) toward endoderm was studied on bFGF-modified PAA brush surfaces. Finally, the results illustrate that PAA brushes, particularly those biofunctionalized with chemisorbed GFs, produce an expected measurable effect on both cell types. Therefore, PAA polymer brushes biofunctionalized with GFs can be used as bioactive cell culture substrates with tuned efficiency.

Heparinization of a biomimetic bone matrix: integration of heparin during matrix synthesis versus adsorptive post surface modification.

This study intended to evaluate a contemporary concept of scaffolding in bone tissue engineering in order to mimic functions of the extracellular matrix. The investigated approach considered the effect of the glycosaminoglycan heparin on structural and biological properties of a synthetic biomimetic bone graft material consisting of mineralized collagen. Two strategies for heparin functionalization were explored in order to receive a three-component bone substitute material. Heparin was either incorporated during matrix synthesis by mixing with collagen prior to simultaneous fibril reassembly and mineralization (in situ) or added to the matrix after fabrication (a posteriori). Both methods resulted in an incorporation of comparable amounts of heparin, though its distribution in the matrix varied as indicated by TOF-SIMS analyses, and a similar modulation of their protein binding properties. Differential scanning calorimetry revealed that the thermal stability and thereby the degree of crosslinking of the heparinized matrices was increased. However, in contrast to the a posteriori modification, the in situ integration of heparin led to considerable changes of morphology and composition of the matrix: a more open network of collagen fibers yielding a more porous surface and a reduced mineral content were observed. Cell culture experiments with human mesenchymal stem cells (hMSC) revealed a strong influence of the mode of heparin functionalization on cellular processes, as demonstrated for proliferation and osteogenic differentiation of hMSC. Our results indicate that not only heparin per se but also the way of its incorporation into a collagenous matrix determines the cell response. In conclusion, the a posteriori modification was beneficial to support adhesion, proliferation and differentiation of hMSC.

Hemocompatibility tuning of an innovative glutaraldehyde-free preparation strategy using riboflavin/UV crosslinking and electron irradiation of bovine pericardium for cardiac substitutes.

Hemocompatibility tuning was adopted to explore and refine an innovative, GA-free preparation strategy combining decellularization, riboflavin/UV crosslinking, and low-energy electron irradiation (SULEEI) procedure. A SULEEI-protocol was established to avoid GA-dependent deterioration that results in insufficient long-term aortic valve bioprosthesis durability. Final SULEEI-pericardium, intermediate steps and GA-fixed reference pericardium were exposed in vitro to fresh human whole blood to elucidate effects of preparation parameters on coagulation and inflammation activation and tissue histology. The riboflavin/UV crosslinking step showed to be less efficient in inactivating extracellular matrix (ECM) protein activity than the GA fixation, leading to tissue-factor mediated blood clotting. Intensifying the riboflavin/UV crosslinking with elevated riboflavin concentration and dextran caused an enhanced activation of the complement system. Yet activation processes induced by the previous protocol steps were quenched with the final electron beam treatment step. An optimized SULEEI protocol was developed using an intense and extended, trypsin-containing decellularization step to inactivate tissue factor and a dextran-free, low riboflavin, high UV crosslinking step. The innovative and improved GA-free SULEEI-preparation protocol results in low coagulant and low inflammatory bovine pericardium for surgical application.

Investigations Into the Suitability of Bacterial Suspensions as Biological Indicators for Low-Energy Electron Irradiation.

Low-energy electron irradiation is an emerging alternative technology for attenuated or complete pathogen inactivation with respect to medical, biotechnological, and pharmaceutical applications. Pathogen inactivation by ionizing radiation depends mainly on the absorbed electron dose. In low-energy electron irradiation processes, determination of the absorbed electron dose is challenging due to the limited, material-dependent penetration depth of the accelerated electrons into the matter. In general, there are established dosimetry systems to evaluate the absorbed dose under dry irradiation conditions. However, there is no system for precise dose monitoring of low-energy irradiation processes in liquids or suspensions so far. Therefore, in this study three different bacterial species were investigated as biological dose indicators, especially in the range of low doses (< 6.5 kGy) in aqueous solutions or suspensions. Escherichia coli, Bacillus subtilis, and Staphylococcus warneri were comparatively evaluated for their suitability as biological dose indicators. Thin homogeneous films of the respective bacterial suspensions were irradiated with increasing doses of low-energy accelerated electrons. The average absorbed dose was determined using a colorimetric dosimeter based on a tetrazolium salt solution. The maximum and minimum absorbed doses were measured with a referenced film dosimeter. Subsequently, the inactivation kinetics was determined in terms of inactivation curves and D10 values. Thus, the minimum inactivation dose of bacterial growth was assessed for E. coli and S. warneri. The effect of irradiation with low-energy accelerated electrons on the growth behavior and activity of the bacteria was studied in more detail using impedance spectroscopy. With increasing irradiation doses growth was delayed.

Infrared Spectroscopic Verification of a α-Helical Collagen Structure in Glutaraldehyde-Free Crosslinked Bovine Pericardium for Cardiac Implants.

The degeneration of heart valve bioprostheses due to calcification processes is caused by the intercalation of calciumhydroxyapatite in pericardium collagen bundles. Variations of the protein secondary structure of biomaterials according to preparation are relevant for this mineralization process and thus the structural characterization of innovative bioprostheses materials is of great importance. The gold standard for prostheses preparation is glutaraldehyde (GA)-fixation of bovine pericardium that adversely promotes calcification. The novel GA-free SULEEI-treatment of bovine pericardium includes decellularization, UV-crosslinking, and electron beam sterilization. The aim of this study is the structural characterization of SULEEI-treated and GA-fixed bovine pericardium. IR spectroscopic imaging combined with multivariate data and curve fit analysis was applied to investigate the amide I and amide II regions of SULEEI-treated and GA-fixed samples. The spectroscopic images of GA-fixed pericardial tissue exhibited a generally high content of amine groups and side chains providing nucleation points for calcification processes. In contrast, in SULEEI-treated tissue, the typical α-helical structure was retained and was supposed to be less prone to deterioration.

Brillouin confocal microscopy to determine biomechanical properties of SULEEI-treated bovine pericardium for application in cardiac surgery.

BACKGROUND: Heart valves are exposed to a highly dynamic environment and underlie high tensile and shear forces during opening and closing. Therefore, analysis of mechanical performance of novel heart valve bioprostheses materials, like SULEEI-treated bovine pericardium, is essential and usually carried out by uniaxial tensile tests. Nevertheless, major drawbacks are the unidirectional strain, which does not reflect the in vivo condition and the deformation of the sample material. An alternative approach for measurement of biomechanical properties is offered by Brillouin confocal microscopy (BCM), a novel, non-invasive and three-dimensional method based on the interaction of light with acoustic waves. OBJECTIVE: BCM is a powerful tool to determine viscoelastic tissue properties and is, for the first time, applied to characterize novel biological graft materials, such as SULEEI-treated bovine pericardium. Therefore, the method has to be validated as a non-invasive alternative to conventional uniaxial tensile tests. METHODS: Vibratome sections of SULEEI-treated bovine pericardium (decellularized, riboflavin/UV-cross-linked and low-energy electron irradiated) as well as native and GA-fixed controls (n = 3) were analyzed by BCM. In addition, uniaxial tensile tests were performed on equivalent tissue samples and Young's modulus as well as length of toe region were analyzed from stress-strain diagrams. The structure of the extracellular matrix (ECM), especially collagen and elastin, was investigated by multiphoton microscopy (MPM). RESULTS: SULEEI-treated pericardium exhibited a significantly higher Brillouin shift and hence higher tissue stiffness in comparison to native and GA-fixed controls (native: 5.6±0.2 GHz; GA: 5.5±0.1 GHz; SULEEI: 6.3±0.1 GHz; n = 3, p < 0.0001). Similarly, a significantly higher Young's modulus was detected in SULEEI-treated pericardia in comparison to native tissue (native: 30.0±10.4 MPa; GA: 31.8±10.7 MPa; SULEEI: 42.1±7.0 MPa; n = 3, p = 0.027). Native pericardia showed wavy and non-directional collagen fibers as well as thin, linear elastin fibers generating a loose matrix. The fibers of GA-fixed and SULEEI-treated pericardium were aligned in one direction, whereat the SULEEI-sample exhibited a much denser matrix. CONCLUSION: BCM is an innovative and non-invasive method to analyze elastic properties of novel pericardial graft materials with special mechanical requirements, like heart valve bioprostheses.

Low Energy Electron Irradiation Is a Potent Alternative to Gamma Irradiation for the Inactivation of (CAR-)NK-92 Cells in ATMP Manufacturing.

Background: With increasing clinical use of NK-92 cells and their CAR-modified derivatives in cancer immunotherapy, there is a growing demand for efficient production processes of these "off-the-shelf" therapeutics. In order to ensure safety and prevent the occurrence of secondary tumors, (CAR-)NK-92 cell proliferation has to be inactivated before transfusion. This is commonly achieved by gamma irradiation. Recently, we showed proof of concept that low energy electron irradiation (LEEI) is a new method for NK-92 inactivation. LEEI has several advantages over gamma irradiation, including a faster reaction time, a more reproducible dose rate and much less requirements on radiation shielding. Here, LEEI was further evaluated as a promising alternative to gamma irradiation yielding cells with highly maintained cytotoxic effector function. Methods: Effectiveness and efficiency of LEEI and gamma irradiation were analyzed using NK-92 and CD123-directed CAR-NK-92 cells. LEE-irradiated cells were extensively characterized and compared to gamma-irradiated cells via flow cytometry, cytotoxicity assays, and comet assays, amongst others. Results: Our results show that both irradiation methods caused a progressive decrease in cell viability and are, therefore, suitable for inhibition of cell proliferation. Notably, the NK-mediated specific lysis of tumor cells was maintained at stable levels for three days post-irradiation, with a trend towards higher activities after LEEI treatment as compared to gamma irradiation. Both gamma irradiation as well as LEEI led to substantial DNA damage and an accumulation of irradiated cells in the G2/M cell cycle phases. In addition, transcriptomic analysis of irradiated cells revealed approximately 12-fold more differentially expressed genes two hours after gamma irradiation, compared to LEEI. Analysis of surface molecules revealed an irradiation-induced decrease in surface expression of CD56, but no changes in the levels of the activating receptors NKp46, NKG2D, or NKp30. Conclusions: The presented data show that LEEI inactivates (CAR-)NK-92 cells as efficiently as gamma irradiation, but with less impact on the overall gene expression. Due to logistic advantages, LEEI might provide a superior alternative for the manufacture of (CAR-)NK-92 cells for clinical application.

In-situ-Investigation of Enzyme Immobilization on Polymer Brushes.

Herein, we report on the use of a combined setup of quartz-crystal microbalance, with dissipation monitoring and spectroscopic ellipsometry, to comprehensively investigate the covalent immobilization of an enzyme to a polymer layer. All steps of the covalent reaction of the model enzyme glucose oxidase with the poly(acrylic acid) brush by carbodiimide chemistry, were monitored in-situ. Data were analyzed using optical and viscoelastic modeling. A nearly complete collapse of the polymer chains was found upon activation of the carboxylic acid groups with N-(3-Dimethylaminopropyl)-N'-ethylcarbodiimide and N-Hydroxysuccinimide. The reaction with the amine groups of the enzyme occurs simultaneously with re-hydration of the polymer layer. Significantly more enzyme was immobilized on the surface compared to physical adsorption at similar conditions, at the same pH. It was found that the pH responsive swelling behavior was almost not affected by the presence of the enzyme.

Bioinspired thermoresponsive nanoscaled coatings: Tailor-made polymer brushes with bioconjugated arginine-glycine-aspartic acid-peptides.

The development of bioengineered surface coatings with stimuli-responsive properties is beneficial for a number of biomedical applications. Environmentally responsive and switchable polymer brush systems have a great potential to create such smart biointerfaces. This study focuses on the bioconjugation of cell-instructive peptides, containing the arginine-glycine-aspartic acid tripeptide sequence (RGD motif), onto well-defined polymer brush films. Herein, the highly tailored end-grafted homo polymer brushes are either composed of the polyelectrolyte poly(acrylic) acid (PAA), providing the reactive carboxyl functionalities, or of the temperature-responsive poly(N-isopropylacrylamide) (PNIPAAm). Of particular interest is the preparation of grafted-to binary brushes using both polymers and their subsequent conversion to RGD-biofunctionalized PNIPAAm-PAA binary brushes by a carbodiimide conjugation method. The bioconjugation process of two linear RGD-peptides Gly-Arg-Gly-Asp-Ser and Gly-Arg-Gly-Asp-Ser-Pro-Lys and one cyclic RGD-peptide cyclo(Arg-Gly-Asp-D-Tyr-Lys) is comparatively investigated by complementary analysis methods. Both techniques, in situ attenuated total reflectance Fourier transform infrared spectroscopy measurements and the in situ spectroscopic ellipsometric analysis, describe changes of the brush surface properties due to biofunctionalization. Besides, the bound RGD-peptide amount is quantitatively evaluated by ellipsometry in comparison to high performance liquid chromatography analysis data. Additionally, molecular dynamic simulations of the RGD-peptides themselves allow a better understanding of the bioconjugation process depending on the peptide properties. The significant influence on the bioconjugation result can be derived, on the one hand, of the polymer brush composition, especially from the PNIPAAm content, and, on the other hand, of the peptide dimension and its reactivity.

In Situ Monitoring of Linear RGD-Peptide Bioconjugation with Nanoscale Polymer Brushes.

Bioinspired materials mimicking the native extracellular matrix environment are promising for biotechnological applications. Particularly, modular biosurface engineering based on the functionalization of stimuli-responsive polymer brushes with peptide sequences can be used for the development of smart surfaces with biomimetic cues. The key aspect of this study is the in situ monitoring and analytical verification of the biofunctionalization process on the basis of three complementary analytical techniques. In situ spectroscopic ellipsometry was used to quantify the amount of chemisorbed GRGDS at both the homopolymer poly(acrylic acid) (PAA) brush and the binary poly(N-isopropylacrylamide) (PNIPAAm)-PAA brushes, which was finally confirmed by an acidic hydrolysis combined with a subsequent reverse-phase high-performance liquid chromatography analysis. In situ attenuated total reflection-Fourier transform infrared spectroscopy provided a step-by-step detection of the biofunctionalization process so that an optimized protocol for the bioconjugation of GRGDS could be identified. The optimized protocol was used to create a temperature-responsive binary brush with a high amount of chemisorbed GRGDS, which is a promising candidate for the temperature-sensitive control of GRGDS presentation in further cell-instructive studies.

Adsorption of enzymes to stimuli-responsive polymer brushes: Influence of brush conformation on adsorbed amount and biocatalytic activity.

Polyelectrolyte brushes can be utilized to immobilize enzymes on macroscopic surfaces. This report investigates the influence of the pH value of the surrounding medium on the amount and the activity of enzymes adsorbed to poly(2-vinylpyridine) and poly(acrylic acid) brushes, as well as the creation of thermoresponsive biocatalytically active coatings via the adsorption of enzymes onto a mixed brush consisting of a polyelectrolyte and temperature-sensitive poly(N-isopropylacryl amide). Spectroscopic ellipsometry and attenuated total reflection-Fourier transform infrared spectroscopy are used to monitor the adsorption process. Additionally, infrared spectra are evaluated in terms of the secondary structure of the enzymes. Glucose oxidase is used as a model enzyme, where the enzymatic activity is measured after different adsorption conditions. Poly(acrylic acid) brushes generally adsorb larger amounts of enzyme, while less glucose oxidase is found on poly(2-vinylpyridine), which however exhibits higher specific activity. This difference in activity could be attributed to a difference in secondary structure of the adsorbed enzyme. For glucose oxidase adsorbed to mixed brushes, switching of enzymatic activity between an active state at 20°C and a less active state at 40°C as compared to the free enzyme in solution is observed. However, this switching is strongly depending on pH in mixed brushes of poly(acrylic acid) and poly(N-isopropylacryl amide) due to interactions between the polymers.

Biomimetic porous scaffolds with high elasticity made from mineralized collagen--an animal study.

Histological investigations of a new hydroxyapatite-collagen composite material were carried out to evaluate its possible suitability as a bone substitute. The three-dimensional scaffolds made from biomimetically mineralized collagen exhibit an interconnecting pore structure and elastic mechanical properties. They were implanted into the subcutaneous tissue and bone defects made in the femur of rats and harvested with the surrounding tissue at 1, 2, 4, 8, and 12 weeks after surgery. The materials implanted in the subcutaneous tissue were covered by fibrous connective tissue with a slight inflammatory response, and many foreign-body giant cells were observed on the surface of the scaffolds. Most of the material implanted in the subcutaneous tissue was resorbed at 8 weeks by phagocytosis. In the bone defects, new bone formation was observed on the surface of the material at 1 week. New bone increased with time, and osteoclasts were seen on the surface of the scaffolds at 2 weeks. Resorption and replacement by new bone of many parts of the materials implanted in the femur were observed by 12 weeks. These responses occurred faster than those of other hydroxyapatite-collagen composites. The results suggested that the new biomimetically mineralized collagen scaffolds were suitable as an implant material for bone-tissue reconstruction.

Nanostructured Biointerfaces: Nanoarchitectonics of Thermoresponsive Polymer Brushes Impact Protein Adsorption and Cell Adhesion.

Controlling the reversibility, quantity, and extent of biomolecule interaction at interfaces has a significant relevance for biomedical and biotechnological applications, because protein adsorption is always the first step when a solid surface gets in contact with a biological fluid. Polymer brushes, composed of end-tethered linear polymers with sufficient grafting density, are very promising to control and alter interactions with biological systems because of their unique structure and distinct collaborative response to environmental changes. We studied protein adsorption and cell adhesion at polymer brush substrates which consisted of poly(N-isopropylacrylamide) (PNIPAAm), having a lower critical solution temperature (LCST), to control bioadsorptive processes by changing the environmental temperature. Preparing the PNIPAAm brushes by the "grafting-to"-method two differently synthesized PNIPAAm polymers were used, at which one possessed an additional hydrophobic terminal headgroup. It is known that hydrophobic moieties can influence protein adsorption significantly. The films were comprehensively analyzed by in situ spectroscopic ellipsometry, contact angle measurements, streaming potential, and atomic force microscopy. Our study was mainly focused on the investigation of the fibrinogen (FGN) adsorption responsiveness both on homo polymer PNIPAAm brushes with and without the hydrophobic terminal functionalization, and further on binary brushes made of the polyelectrolyte poly(acrylic acid) (PAA) and one of the prior described two PNIPAAm species. The results show that the terminal hydrophobic modification of PNIPAAm has a considerable impact on wettability, LCST, and morphology of the homo and the binary brush systems, which consequently led to an alteration of FGN adsorption. By using binary PNIPAAm-PAA brushes with different composition it was possible to induce stimuli dependent FGN adsorption with a considerable amplified switching effect by introducing a hydrophobic terminal residue to PNIPAAm. Cell adhesion studies with human mesenchymal stem cells reflected the results of the FGN adsorption.

Cages augmented with mineralized collagen and platelet-rich plasma as an osteoconductive/inductive combination for interbody fusion.

STUDY DESIGN: After anterior cervical discectomy, fusion was radiologically, biomechanically, and histologically assessed in a sheep spine fusion model. OBJECTIVE: To evaluate the efficacy of a platelet-rich plasma (PRP) application combined with a mineralized collagen matrix (MCM) as an alternative to autologous cancellous iliac crest bone grafts in a spine fusion model. SUMMARY OF BACKGROUND DATA: PRP has the ability to stimulate bone and tissue healing. MCM is a recently developed osteoconductive material. Up to now, no comparative evaluation of PRP in combination with a MCM at the cervical spine has been performed in vivo. METHODS: Twenty-four sheep (N = 8/group) underwent C3/4 discectomy and fusion: group 1, titanium cage filled with autologous cancellous iliac crest bone graft; group 2, titanium cage filled with MCM; and group 3, titanium cage filled with MCM and PRP. Radiographic evaluation was performed before surgery and after 1, 2, 4, 8, and 12 weeks, respectively. After 12 weeks, fusion sites were evaluated using functional radiographic views and quantitative computed tomographic scans to assess bone mineral density. Furthermore, histomorphologic and histomorphometrical analyses were performed to evaluate fusion. RESULTS: In comparison with the titanium cage group filled with autologous cancellous iliac crest bone grafts representing the control group, MCM-alone group showed a slightly lower fusion rate in the radiographic and the histomorphometrical analysis. The addition of PRP could not enhance this finding. There was no significant difference between MCM and MCM + PRP group in radiologic and histologic findings. CONCLUSION: The MCM alone is not able to replace autologous bone grafts. Early activation of the platelets by calcium, which is released from mineralized collagen, could be the reason for the insufficient osteoinductive effect of PRP. In consequence, the combined application of mineralized collagen and PRP had no significant osteoinductive effect in this model.