Suitability of R. pulmo Jellyfish-Collagen-Coated Well Plates for Cytocompatibility Analyses of Biomaterials.

Cytocompatibility analyses of new implant materials or biomaterials are not only prescribed by the Medical Device Regulation (MDR), as defined in the DIN ISO Norm 10993-5 and -12, but are also increasingly replacing animal testing. In this context, jellyfish collagen has already been established as an alternative to mammalian collagen in different cell culture conditions, but a lack of knowledge exists about its applicability for cytocompatibility analyses of biomaterials. Thus, the present study was conducted to compare well plates coated with collagen type 0 derived from Rhizostoma pulmo with plates coated with bovine and porcine collagen. The coated well plates were analysed in vitro for their cytocompatibility, according to EN ISO 10993-5/-12, using both L929 fibroblasts and MC3T3 pre-osteoblasts. Thereby, the coated well plates were compared, using established materials as positive controls and a cytotoxic material, RM-A, as a negative control. L929 cells exhibited a significantly higher viability (#### p < 0.0001), proliferation (## p < 0.01), and a lower cytotoxicity (## p < 0.01 and # p < 0.05)) in the Jellagen® group compared to the bovine and porcine collagen groups. MC3T3 cells showed similar viability and acceptable proliferation and cytotoxicity in all collagen groups. The results of the present study revealed that the coating of well plates with collagen Type 0 derived from R. pulmo leads to comparable results to the case of well plates coated with mammalian collagens. Therefore, it is fully suitable for the in vitro analyses of the cytocompatibility of biomaterials or medical devices.

The Use of Collagen-Based Materials in Bone Tissue Engineering.

Synthetic bone substitute materials (BSMs) are becoming the general trend, replacing autologous grafting for bone tissue engineering (BTE) in orthopedic research and clinical practice. As the main component of bone matrix, collagen type I has played a critical role in the construction of ideal synthetic BSMs for decades. Significant strides have been made in the field of collagen research, including the exploration of various collagen types, structures, and sources, the optimization of preparation techniques, modification technologies, and the manufacture of various collagen-based materials. However, the poor mechanical properties, fast degradation, and lack of osteoconductive activity of collagen-based materials caused inefficient bone replacement and limited their translation into clinical reality. In the area of BTE, so far, attempts have focused on the preparation of collagen-based biomimetic BSMs, along with other inorganic materials and bioactive substances. By reviewing the approved products on the market, this manuscript updates the latest applications of collagen-based materials in bone regeneration and highlights the potential for further development in the field of BTE over the next ten years.

Analyses of the Cellular Interactions between the Ossification of Collagen-Based Barrier Membranes and the Underlying Bone Defects.

Barrier membranes are an essential tool in guided bone Regeneration (GBR), which have been widely presumed to have a bioactive effect that is beyond their occluding and space maintenance functionalities. A standardized calvaria implantation model was applied for 2, 8, and 16 weeks on Wistar rats to test the interactions between the barrier membrane and the underlying bone defects which were filled with bovine bone substitute materials (BSM). In an effort to understand the barrier membrane's bioactivity, deeper histochemical analyses, as well as the immunohistochemical detection of macrophage subtypes (M1/M2) and vascular endothelial cells, were conducted and combined with histomorphometric and statistical approaches. The native collagen-based membrane was found to have ossified due to its potentially osteoconductive and osteogenic properties, forming a "bony shield" overlying the bone defects. Histomorphometrical evaluation revealed the resorption of the membranes and their substitution with bone matrix. The numbers of both M1- and M2-macrophages were significantly higher within the membrane compartments compared to the underlying bone defects. Thereby, M2-macrophages significantly dominated the tissue reaction within the membrane compartments. Statistically, a correlation between M2-macropahges and bone regeneration was only found at 2 weeks post implantationem, while the pro-inflammatory limb of the immune response correlated with the two processes at 8 weeks. Altogether, this study elaborates on the increasingly described correlations between barrier membranes and the underlying bone regeneration, which sheds a light on the understanding of the immunomodulatory features of biomaterials.

Comparative analysis of lateral maxillary sinus augmentation with a xenogeneic bone substitute material in combination with piezosurgical preparation and bony wall repositioning or rotary instrumentation and membrane coverage: a prospective randomized clinical and histological study.

OBJECTIVES: The present randomized controlled clinical study aimed to investigate if, in lateral maxillary sinus augmentation, the repositioned bony wall or the application of a collagen membrane results in more preferable new hard tissue formation. MATERIALS AND METHODS: Forty patients were divided into two study groups. Both groups received a xenogeneic bone substitute material (BSM) during lateral sinus augmentation. In the bony wall group (BW), following piezosurgery, the retrieved bony wall was repositioned. In the collagen membrane group (CM), following rotary instrument preparation, collagen membrane coverage was applied. After 6 months, biopsies were taken to histologically analyze the percentage of BSM, connective tissue (CT), and newly formed bone (NFB) following both approaches. RESULTS: Forty implants were placed and 29 harvested biopsies could be evaluated. Duration of surgery, membrane perforations, and VAS were detected. Histomorphometrical analysis revealed comparable amounts of all analyzed parameters in both groups in descending order: CT (BW: 39.2 ± 9%, CM: 37,9 ± 8.5%) > BSM (BW: 32.9 ± 6.3%, CM: 31.8 ± 8.8%) > NB (BW: 27.8 ± 11.2%, CM: 30.3 ± 4.5%). CONCLUSIONS: The results of the present study show that the closure of the access window by means of the retrieved bony wall or a native collagen membrane led to comparable bone augmentation results. CLINICAL TRIAL: clinicaltrials.gov NCT04811768. CLINICAL RELEVANCE: Lateral maxillary sinus augmentation with the application of a xenogeneic BSM in combination with a native collagen membrane for bony window coverage represents a reliable method for surgical reconstruction of the posterior maxilla. Piezosurgery with bony window repositioning delivers comparable outcomes without membrane coverage.

In Vivo Biocompatibility Investigation of an Injectable Calcium Carbonate (Vaterite) as a Bone Substitute including Compositional Analysis via SEM-EDX Technology.

Injectable bone substitutes (IBS) are increasingly being used in the fields of orthopedics and maxillofacial/oral surgery. The rheological properties of IBS allow for proper and less invasive filling of bony defects. Vaterite is the most unstable crystalline polymorph of calcium carbonate and is known to be able to transform into hydroxyapatite upon contact with an organic fluid (e.g., interstitial body fluid). Two different concentrations of hydrogels based on poly(ethylene glycol)-acetal-dimethacrylat (PEG-a-DMA), i.e., 8% (w/v) (VH-A) or 10% (w/v) (VH-B), were combined with vaterite nanoparticles and implanted in subcutaneous pockets of BALB/c mice for 15 and 30 days. Explants were prepared for histochemical staining and immunohistochemical detection methods to determine macrophage polarization, and energy-dispersive X-ray analysis (EDX) to analyze elemental composition was used for the analysis. The histopathological analysis revealed a comparable moderate tissue reaction to the hydrogels mainly involving macrophages. Moreover, the hydrogels underwent a slow cellular infiltration, revealing a different degradation behavior compared to other IBS. The immunohistochemical detection showed that M1 macrophages were mainly found at the material surfaces being involved in the cell-mediated degradation and tissue integration, while M2 macrophages were predominantly found within the reactive connective tissue. Furthermore, the histomorphometrical analysis revealed balanced numbers of pro- and anti-inflammatory macrophages, demonstrating that both hydrogels are favorable materials for bone tissue regeneration. Finally, the EDX analysis showed a stepwise transformation of the vaterite particle into hydroxyapatite. Overall, the results of the present study demonstrate that hydrogels including nano-vaterite particles are biocompatible and suitable for bone tissue regeneration applications.

Barrier Membranes for Guided Bone Regeneration (GBR): A Focus on Recent Advances in Collagen Membranes.

Guided bone regeneration (GBR) has become a clinically standard modality for the treatment of localized jawbone defects. Barrier membranes play an important role in this process by preventing soft tissue invasion outgoing from the mucosa and creating an underlying space to support bone growth. Different membrane types provide different biological mechanisms due to their different origins, preparation methods and structures. Among them, collagen membranes have attracted great interest due to their excellent biological properties and desired bone regeneration results to non-absorbable membranes even without a second surgery for removal. This work provides a comparative summary of common barrier membranes used in GBR, focusing on recent advances in collagen membranes and their biological mechanisms. In conclusion, the review article highlights the biological and regenerative properties of currently available barrier membranes with a particular focus on bioresorbable collagen-based materials. In addition, the advantages and disadvantages of these biomaterials are highlighted, and possible improvements for future material developments are summarized.

In Vivo Analysis of the Regeneration Capacity and Immune Response to Xenogeneic and Synthetic Bone Substitute Materials.

Although various studies have investigated differences in the tissue reaction pattern to synthetic and xenogeneic bone substitute materials (BSMs), a lack of knowledge exists regarding the classification of both materials based on the DIN ISO 10993-6 scoring system, as well as the histomorphometrical measurement of macrophage subtypes within their implantation beds. Thus, the present study was conducted to analyze in vivo responses to both xenogeneic and synthetic bone substitute granules. A standardized calvaria implantation model in Wistar rats, in combination with established scoring, histological, histopathological, and histomorphometrical methods, was conducted to analyze the influence of both biomaterials on bone regeneration and the immune response. The results showed that the application of the synthetic BSM maxresorb® induced a higher pro-inflammatory tissue response, while the xenogeneic BSM cerabone® induced a higher anti-inflammatory reaction. Additionally, comparable bone regeneration amounts were found in both study groups. Histopathological scoring revealed that the synthetic BSM exhibited non-irritant scores at all timepoints using the xenogeneic BSM as control. Overall, the results demonstrated the biocompatibility of synthetic BSM maxresorb® and support the conclusion that this material class is a suitable alternative to natural BSM, such as the analyzed xenogeneic material cerabone®, for a broad range of indications.

Exploring the Biomaterial-Induced Secretome: Physical Bone Substitute Characteristics Influence the Cytokine Expression of Macrophages.

In addition to their chemical composition various physical properties of synthetic bone substitute materials have been shown to influence their regenerative potential and to influence the expression of cytokines produced by monocytes, the key cell-type responsible for tissue reaction to biomaterials in vivo. In the present study both the regenerative potential and the inflammatory response to five bone substitute materials all based on ß-tricalcium phosphate (ß-TCP), but which differed in their physical characteristics (i.e., granule size, granule shape and porosity) were analyzed for their effects on monocyte cytokine expression. To determine the effects of the physical characteristics of the different materials, the proliferation of primary human osteoblasts growing on the materials was analyzed. To determine the immunogenic effects of the different materials on human peripheral blood monocytes, cells cultured on the materials were evaluated for the expression of 14 pro- and anti-inflammatory cytokines, i.e., IL-6, IL-10, IL-1ß, VEGF, RANTES, IL-12p40, I-CAM, IL-4, V-CAM, TNF-α, GM-CSF, MIP-1α, Il-8 and MCP-1 using a Bio-Plex® Multiplex System. The granular shape of bone substitutes showed a significant influence on the osteoblast proliferation. Moreover, smaller pore sizes, round granular shape and larger granule size increased the expression of GM-CSF, RANTES, IL-10 and IL-12 by monocytes, while polygonal shape and the larger pore sizes increased the expression of V-CAM. The physical characteristics of a bone biomaterial can influence the proliferation rate of osteoblasts and has an influence on the cytokine gene expression of monocytes in vitro. These results indicate that the physical structure of a biomaterial has a significant effect of how cells interact with the material. Thus, specific characteristics of a material may strongly affect the regenerative potential in vivo.

In Vivo Analysis of the Biocompatibility and Bone Healing Capacity of a Novel Bone Grafting Material Combined with Hyaluronic Acid.

The present in vivo study analyses both the inflammatory tissue reactions and the bone healing capacity of a newly developed bone substitute material (BSM) based on xenogeneic bone substitute granules combined with hyaluronate (HY) as a water-binding molecule. The results of the hyaluronate containing bone substitute material (BSM) were compared to a control xenogeneic BSM of the same chemical composition and a sham operation group up to 16 weeks post implantationem. A major focus of the study was to analyze the residual hyaluronate and its effects on the material-dependent healing behavior and the inflammatory tissue responses. The study included 63 male Wistar rats using the calvaria implantation model for 2, 8, and 16 weeks post implantationem. Established and Good Laboratory Practice (GLP)-conforming histological, histopathological, and histomorphometrical analysis methods were conducted. The results showed that the new hyaluronate containing BSM was gradually integrated within newly formed bone up to the end of the study that ended in a condition of complete bone defect healing. Thereby, no differences to the healing capacity of the control BSM were found. However, the bone formation in both groups was continuously significantly higher compared to the sham operation group. Additionally, no differences in the (inflammatory) tissue response that was analyzed via qualitative and (semi-) quantitative methods were found. Interestingly, no differences were found between the numbers of pro- and anti-inflammatory macrophages between the three study groups over the entire course of the study. No signs of the HY as a water-binding part of the BSM were histologically detectable at any of the study time points, altogether the results of the present study show that HY allows for an optimal material-associated bone tissue healing comparable to the control xenogeneic BSM. The added HY seems to be degraded within a very short time period of less than 2 weeks so that the remaining BSM granules allow for a gradual osteoconductive bone regeneration. Additionally, no differences between the inflammatory tissue reactions in both material groups and the sham operation group were found. Thus, the new hyaluronate containing xenogeneic BSM and also the control BSM have been shown to be fully biocompatible without any differences regarding bone regeneration.

Ex Vivo and In Vivo Analysis of a Novel Porcine Aortic Patch for Vascular Reconstruction.

(1) Background: The aim of the present study was the biocompatibility analysis of a novel xenogeneic vascular graft material (PAP) based on native collagen won from porcine aorta using the subcutaneous implantation model up to 120 days post implantationem. As a control, an already commercially available collagen-based vessel graft (XenoSure®) based on bovine pericardium was used. Another focus was to analyze the (ultra-) structure and the purification effort. (2) Methods: Established methodologies such as the histological material analysis and the conduct of the subcutaneous implantation model in Wistar rats were applied. Moreover, established methods combining histological, immunohistochemical, and histomorphometrical procedures were applied to analyze the tissue reactions to the vessel graft materials, including the induction of pro- and anti-inflammatory macrophages to test the immune response. (3) Results: The results showed that the PAP implants induced a special cellular infiltration and host tissue integration based on its three different parts based on the different layers of the donor tissue. Thereby, these material parts induced a vascularization pattern that branches to all parts of the graft and altogether a balanced immune tissue reaction in contrast to the control material. (4) Conclusions: PAP implants seemed to be advantageous in many aspects: (i) cellular infiltration and host tissue integration, (ii) vascularization pattern that branches to all parts of the graft, and (iii) balanced immune tissue reaction that can result in less scar tissue and enhanced integrative healing patterns. Moreover, the unique trans-implant vascularization can provide unprecedented anti-infection properties that can avoid material-related bacterial infections.

Ex Vivo and In Vivo Analyses of Novel 3D-Printed Bone Substitute Scaffolds Incorporating Biphasic Calcium Phosphate Granules for Bone Regeneration.

(1) Background: The aim of this study was examining the ex vivo and in vivo properties of a composite made from polycaprolactone (PCL) and biphasic calcium phosphate (BCP) (synprint, ScientiFY GmbH) fabricated via fused deposition modelling (FDM); (2) Methods: Scaffolds were tested ex vivo for their mechanical properties using porous and solid designs. Subcutaneous implantation model analyzed the biocompatibility of PCL + BCP and PCL scaffolds. Calvaria implantation model analyzed the osteoconductive properties of PCL and PCL + BCP scaffolds compared to BCP as control group. Established histological, histopathological and histomorphometrical methods were performed to evaluate new bone formation.; (3) Results Mechanical testing demonstrated no significant differences between PCL and PCL + BCP for both designs. Similar biocompatibility was observed subcutaneously for PCL and PCL + BCP scaffolds. In the calvaria model, new bone formation was observed for all groups with largest new bone formation in the BCP group, followed by the PCL + BCP group, and the PCL group. This finding was influenced by the initial volume of biomaterial implanted and remaining volume after 90 days. All materials showed osteoconductive properties and PCL + BCP tailored the tissue responses towards higher cellular biodegradability. Moreover, this material combination led to a reduced swelling in PCL + BCP; (4) Conclusions: Altogether, the results show that the newly developed composite is biocompatible and leads to successful osteoconductive bone regeneration. The new biomaterial combines the structural stability provided by PCL with bioactive characteristics of BCP-based BSM. 3D-printed BSM provides an integration behavior in accordance with the concept of guided bone regeneration (GBR) by directing new bone growth for proper function and restoration.

Biocompatibility Analyses of HF-Passivated Magnesium Screws for Guided Bone Regeneration (GBR).

Background: Magnesium (Mg) is one of the most promising materials for human use in surgery due to material characteristics such as its elastic modulus as well as its resorbable and regenerative properties. In this study, HF-coated and uncoated novel bioresorbable magnesium fixation screws for maxillofacial and dental surgical applications were investigated in vitro and in vivo to evaluate the biocompatibility of the HF coating. Methods: Mg alloy screws that had either undergone a surface treatment with hydrofluoric-acid (HF) or left untreated were investigated. In vitro investigation included XTT, BrdU and LDH in accordance with the DIN ISO 10993-5/-12. In vivo, the screws were implanted into the tibia of rabbits. After 3 and 6 weeks, degradation, local tissue reactions and bony integration were analyzed histopathologically and histomorphometrically. Additionally, SEM/EDX analysis and synchrotron phase-contrast microtomography (µCT) measurements were conducted. The in vitro analyses revealed that the Mg screws are cytocompatible, with improved results when the surface had been passivated with HF. In vivo, the HF-treated Mg screws implanted showed a reduction in gas formation, slower biodegradation and a better bony integration in comparison to the untreated Mg screws. Histopathologically, the HF-passivated screws induced a layer of macrophages as part of its biodegradation process, whereas the untreated screws caused a slight fibrous tissue reaction. SEM/EDX analysis showed that both screws formed a similar layer of calcium phosphates on their surfaces and were surrounded by bone. Furthermore, the µCT revealed the presence of a metallic core of the screws, a faster absorbing corrosion front and a slow absorbing region of corroded magnesium. Conclusions: Overall, the HF-passivated Mg fixation screws showed significantly better biocompatibility in vitro and in vivo compared to the untreated screws.

In Vivo Analysis of the Biocompatibility and Immune Response of Jellyfish Collagen Scaffolds and its Suitability for Bone Regeneration.

Jellyfish collagen, which can be defined as "collagen type 0" due to its homogeneity to the mammalian types I, II, III, V, and IX and its batch-to-batch consistent producibility, is of special interest for different medical applications related to (bone) tissue regeneration as an alternative to mammalian collagen-based biomaterials. However, no in vivo studies regarding the induction of M1- and M2-macrophages and their time-dependent ration as well as the analysis of the bone regeneration capacity of jellyfish collagen scaffolds have been conducted until now. Thus, the goal of this study was to determine the nature of the immune response to jellyfish collagen scaffolds and their bone healing capacities. Two in vivo studies using established implantation models, i.e., the subcutaneous and the calvarian implantation model in Wistar rats, were conducted. Furthermore, specialized histological, histopathological, and histomorphometrical methods have been used. As a control biomaterial, a collagen scaffold, originating from porcine pericardium, which has already been stated as biocompatible, was used for the subcutaneous study. The results of the present study show that jellyfish collagen scaffolds are nearly completely resorbed until day 60 post implantation by stepwise integration within the subcutaneous connective tissue mediated mainly by macrophages and single multinucleated giant cells. Interestingly, the degradation process ended in a vessel rich connective tissue that is understood to be an optimal basis for tissue regeneration. The study results showed an overall weaker immune response to jellyfish collagen than to porcine pericardium matrices by the induction of significantly lower numbers of macrophages together with a more balanced occurrence of M1- and M2-macrophages. However, both collagen-based biomaterials induced balanced numbers of both macrophage subtypes, which supports their good biocompatibility. Moreover, the histomorphometrical results for the calvarial implantation of the jellyfish scaffolds revealed an average of 46.20% de novo bone formation at day 60, which was significantly higher compared to the control group. Thereby, the jellyfish collagen scaffolds induced also significantly higher numbers of anti-inflammatory macrophages within the bony implantation beds. Altogether, the results show that the jellyfish collagen scaffolds allowed for a directed integration behavior, which is assumed to be in accordance with the concept of Guided Bone Regeneration (GBR). Furthermore, the jellyfish collagen scaffolds induced a long-term anti-inflammatory macrophage response and an optimal vascularization pattern within their implant beds, thus showing excellent biocompatibility and (bone) tissue healing properties.

Degradation, Bone Regeneration and Tissue Response of an Innovative Volume Stable Magnesium-Supported GBR/GTR Barrier Membrane.

INTRODUCTION: Bioresorbable collagenous barrier membranes are used to prevent premature soft tissue ingrowth and to allow bone regeneration. For volume stable indications, only non-absorbable synthetic materials are available. This study investigates a new bioresorbable hydrofluoric acid (HF)-treated magnesium (Mg) mesh in a native collagen membrane for volume stable situations. MATERIALS AND METHODS: HF-treated and untreated Mg were compared in direct and indirect cytocompatibility assays. In vivo, 18 New Zealand White Rabbits received each four 8 mm calvarial defects and were divided into four groups: (a) HF-treated Mg mesh/collagen membrane, (b) untreated Mg mesh/collagen membrane (c) collagen membrane and (d) sham operation. After 6, 12 and 18 weeks, Mg degradation and bone regeneration was measured using radiological and histological methods. RESULTS: In vitro, HF-treated Mg showed higher cytocompatibility. Histopathologically, HF-Mg prevented gas cavities and was degraded by mononuclear cells via phagocytosis up to 12 weeks. Untreated Mg showed partially significant more gas cavities and a fibrous tissue reaction. Bone regeneration was not significantly different between all groups. DISCUSSION AND CONCLUSIONS: HF-Mg meshes embedded in native collagen membranes represent a volume stable and biocompatible alternative to the non-absorbable synthetic materials. HF-Mg shows less corrosion and is degraded by phagocytosis. However, the application of membranes did not result in higher bone regeneration.

A low-speed centrifugation concept leads to cell accumulation and vascularization of solid platelet-rich fibrin: an experimental study in vivo.

Platelet-rich fibrin (PRF) is generated from the patients' own venous blood by a single centrifugation step without the additional use of anticoagulants. Based on the previously described LSCC (low-speed centrifugation concept), our group showed that modification of the centrifugation setting, that is, reducing the relative centrifugal force (RCF) and mildly increasing the centrifugation time, resulted in modified solid and liquid PRF-matrices with increased number of platelets, leukocytes, and growth factors' concentrations. The aim of this study was to determine whether RCF reduction might also result in different tissue reactions toward the two PRF-based matrices, especially vascularization and cell distribution in vivo. Two centrifugation protocols (PRF-high [719 g] and PRF-medium [222 g]) were compared in a subcutaneous implantation model of SCID mice at 5 and 10 days. Histological and histomorphometrical analyses were performed to quantify lymphocyte, neutrophil, human macrophage, and monocyte populations. CD31 was used to detect newly formed vessels, while all human cells were detected by using human vimentin as a pan-cellular marker. The results demonstrated that PRF-high elicited a dense and stable fibrin structure and prevented cellular penetration of the host tissue. By contrast, PRF-medium was more porous, had a significantly higher in vivo vascularization rate, and included significantly more human cells, especially at day 10, compared to PRF-high. These findings highlight the possibility of modifying the structure and composition of PRF matrices and thus selectively altering their regenerative potential in vivo. Clinical studies now must evaluate the different PRF matrices for bone and soft-tissue regeneration to validate possible benefits using personalized preparation protocols.

Cytocompatibility of Titanium, Zirconia and Modified PEEK after Surface Treatment Using UV Light or Non-Thermal Plasma.

A number of modifications have been developed in order to enhance surface cytocompatibility for prosthetic support of dental implants. Among them, ultraviolet (UV) light and non-thermal plasma (NTP) treatment are promising methods. The objective of this study was to compare the effects of UV light and NTP on machined titanium, zirconia and modified polyetheretherketone (PEEK, BioHPP) surfaces in vitro. Machined samples of titanium, zirconia and BioHPP were treated by UV light and NTP of argon or oxygen for 12 min each. Non-treated disks were set as controls. A mouse fibroblast and a human gingival fibroblast cell line were used for in vitro experiments. After 2, 24 and 48 h of incubation, the attachment, viability and cytotoxicity of cells on surfaces were assessed. Results: Titanium, zirconia and BioHPP surfaces treated by UV light and oxygen plasma were more favorable to the early attachment of soft-tissue cells than non-treated surfaces, and the number of cells on those treated surfaces was significantly increased after 2, 24 and 48 h of incubation (p < 0.05). However, the effects of argon plasma treatment on the cytocompatibility of soft tissue cells varied with the type of cells and the treated material. UV light and oxygen plasma treatments may improve the attachment of fibroblast cells on machined titanium, zirconia and PEEK surfaces, that are materials for prosthetic support of dental implants.

The Addition of High Doses of Hyaluronic Acid to a Biphasic Bone Substitute Decreases the Proinflammatory Tissue Response.

Biphasic bone substitutes (BBS) are currently well-established biomaterials. Through their constant development, even natural components like hyaluronic acid (HY) have been added to improve both their handling and also their regenerative properties. However, little knowledge exists regarding the consequences of the addition of HY to their biocompatibility and the inflammatory tissue reactions. Thus, the present study was conducted, aiming to analyze the influence of two different amounts of high molecular weight HY (HMWHY), combined with a BBS, on in vitro biocompatibility and in vivo tissue reaction. Established in vitro procedures, using L929 cells, were used for cytocompatibility analyses under the test conditions of DIN EN:ISO 10993-5. For the in vivo part of the study, calvarial defects were created in 20 Wistar rats and subsequently filled with BBS, and BBS combined with two different HMWHY amounts, i.e., BBS + HY(L) and BBS + HY(H). As controls, empty defects were used. Established histological, immunohistochemical, and histomorphometrical methods were applied to analyze the tissue reactions to the three different materials, including the induction of pro- and anti-inflammatory macrophages and multinucleated giant cells (BMGCs). The in vitro results showed that none of the materials or compositions caused biological damage to the L929 cells and can be considered to be non-toxic. The in vivo results showed that only the addition of high doses of HY to a biphasic bone substitute significantly decreases the occurrence of pro-inflammatory macrophages (* p < 0.05), comparable to the numbers found in the control group, while no significant differences within the three study groups for M2-macrophages nor BMGCs were detected. In conclusion, the addition of different amounts of HMWHY does not seem to affect the inflammation response to BBS, while improving the material handling properties.

In Vivo Simulation of Magnesium Degradability Using a New Fluid Dynamic Bench Testing Approach.

The degradation rate of magnesium (Mg) alloys is a key parameter to develop Mg-based biomaterials and ensure in vivo-mechanical stability as well as to minimize hydrogen gas production, which otherwise can lead to adverse effects in clinical applications. However, in vitro and in vivo results of the same material often differ largely. In the present study, a dynamic test bench with several single bioreactor cells was constructed to measure the volume of hydrogen gas which evolves during magnesium degradation to indicate the degradation rate in vivo. Degradation medium comparable with human blood plasma was used to simulate body fluids. The media was pumped through the different bioreactor cells under a constant flow rate and 37 °C to simulate physiological conditions. A total of three different Mg groups were successively tested: Mg WE43, and two different WE43 plasma electrolytically oxidized (PEO) variants. The results were compared with other methods to detect magnesium degradation (pH, potentiodynamic polarization (PDP), cytocompatibility, SEM (scanning electron microscopy)). The non-ceramized specimens showed the highest degradation rates and vast standard deviations. In contrast, the two PEO samples demonstrated reduced degradation rates with diminished standard deviation. The pH values showed above-average constant levels between 7.4-7.7, likely due to the constant exchange of the fluids. SEM revealed severe cracks on the surface of WE43 after degradation, whereas the ceramized surfaces showed significantly decreased signs of corrosion. PDP results confirmed the improved corrosion resistance of both PEO samples. While WE43 showed slight toxicity in vitro, satisfactory cytocompatibility was achieved for the PEO test samples. In summary, the dynamic test bench constructed in this study enables reliable and simple measurement of Mg degradation to simulate the in vivo environment. Furthermore, PEO treatment of magnesium is a promising method to adjust magnesium degradation.

Improved In Vitro Test Procedure for Full Assessment of the Cytocompatibility of Degradable Magnesium Based on ISO 10993-5/-12.

Magnesium (Mg)-based biomaterials are promising candidates for bone and tissue regeneration. Alloying and surface modifications provide effective strategies for optimizing and tailoring their degradation kinetics. Nevertheless, biocompatibility analyses of Mg-based materials are challenging due to its special degradation mechanism with continuous hydrogen release. In this context, the hydrogen release and the related (micro-) milieu conditions pretend to strictly follow in vitro standards based on ISO 10993-5/-12. Thus, special adaptions for the testing of Mg materials are necessary, which have been described in a previous study from our group. Based on these adaptions, further developments of a test procedure allowing rapid and effective in vitro cytocompatibility analyses of Mg-based materials based on ISO 10993-5/-12 are necessary. The following study introduces a new two-step test scheme for rapid and effective testing of Mg. Specimens with different surface characteristics were produced by means of plasma electrolytic oxidation (PEO) using silicate-based and phosphate-based electrolytes. The test samples were evaluated for corrosion behavior, cytocompatibility and their mechanical and osteogenic properties. Thereby, two PEO ceramics could be identified for further in vivo evaluations.

Cellular Mechanisms Responsible for Success and Failure of Bone Substitute Materials.

Bone grafts, i.e., autologous, allogeneic or synthetic bone substitute materials play an increasing role in reconstructive orthopedic surgery. While the indications and materials differ, it is important to understand the cellular mechanisms regarding their integration and remodeling, which are discussed in this review article. Osteoconductivity describes the new bone growth on the graft, while osteoinductivity represents the differentiation of undifferentiated cells into bone forming osteoblasts. The best case is that both mechanisms are accompanied by osteogenesis, i.e., bone modeling and remodeling of the graft material. Graft incorporation is mediated by a number of molecular pathways that signal the differentiation and activity of osteoblasts and osteoclasts (e.g., parathyroid hormone (PTH) and receptor activator of nuclear factor κß ligand (RANKL), respectively). Direct contact of the graft and host bone as well as the presence of a mechanical load are a prerequisite for the successful function of bone grafts. Interestingly, while bone substitutes show good to excellent clinical outcomes, their histological incorporation has certain limits that are not yet completely understood. For instance, clinical studies have shown contrasting results regarding the complete or incomplete resorption and remodeling of allografts and synthetic grafts. In this context, a foreign body response can lead to complete material degradation via phagocytosis, however it may also cause a fibrotic reaction to the bone substitute. Finally, the success of bone graft incorporation is also limited by other factors, including the bone remodeling capacities of the host, the material itself (e.g., inadequate resorption, toxicity) and the surgical technique or preparation of the graft.