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.

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.

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 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.

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.

Additive Manufacturing for Guided Bone Regeneration: A Perspective for Alveolar Ridge Augmentation.

Three-dimensional (3D) printing has become an important tool in the field of tissue engineering and its further development will lead to completely new clinical possibilities. The ability to create tissue scaffolds with controllable characteristics, such as internal architecture, porosity, and interconnectivity make it highly desirable in comparison to conventional techniques, which lack a defined structure and repeatability between scaffolds. Furthermore, 3D printing allows for the production of scaffolds with patient-specific dimensions using computer-aided design. The availability of commercially available 3D printed permanent implants is on the rise; however, there are yet to be any commercially available biodegradable/bioresorbable devices. This review will compare the main 3D printing techniques of: stereolithography; selective laser sintering; powder bed inkjet printing and extrusion printing; for the fabrication of biodegradable/bioresorbable bone tissue scaffolds; and, discuss their potential for dental applications, specifically augmentation of the alveolar ridge.

Biphasic bone substitutes coated with PLGA incorporating therapeutic ions Sr2+ and Mg2+: cytotoxicity cascade and in vivo response of immune and bone regeneration.

The incorporation of bioactive ions into biomaterials has gained significant attention as a strategy to enhance bone tissue regeneration on the molecular level. However, little knowledge exists about the effects of the addition of these ions on the immune response and especially on the most important cellular regulators, the macrophages. Thus, this study aimed to investigate the in vitro cytocompatibility and in vivo regulation of bone remodeling and material-related immune responses of a biphasic bone substitute (BBS) coated with metal ions (Sr2+/Mg2+) and PLGA, using the pure BBS as control group. Initially, two cytocompatible modified material variants were identified according to the in vitro results obtained following the DIN EN ISO 10993-5 protocol. The surface structure and ion release of both materials were characterized using SEM-EDX and ICP-OES. The materials were then implanted into Wistar rats for 10, 30, and 90 days using a cranial defect model. Histopathological and histomorphometrical analyses were applied to evaluate material degradation, bone regeneration, osteoconductivity, and immune response. The findings revealed that in all study groups comparable new bone formation were found. However, during the early implantation period, the BBS_Sr2+ group exhibited significantly faster regeneration compared to the other two groups. Additionally, all materials induced comparable tissue and immune responses involving high numbers of both pro-inflammatory macrophages and multinucleated giant cells (MNGCs). In conclusion, this study delved into the repercussions of therapeutic ion doping on bone regeneration patterns and inflammatory responses, offering insights for the advancement of a new generation of biphasic calcium phosphate materials with potential clinical applicability.

Editorial of the Special Issue: "Soft and Hard Tissue Regeneration".

In general, only a total of four tissue classes are distinguished: the covering tissue (epithelial tissue), the connective and supporting tissue (connective tissue, fatty tissue, bone, and cartilage), the muscle tissue, and the nervous tissue [...].

In Vitro Analysis of the Cytocompatibility of a Novel Porcine Aortic Patch for Vascular Reconstruction.

BACKGROUND/AIM: Cardiovascular diseases are one of the most common causes of morbidity and mortality in the world. In the case of severe arteriosclerotic damage, surgical treatment is necessary. Although the use of autologous vessels is still considered to be the gold standard, sufficient autologous vessels for transplantation are lacking. MATERIALS AND METHODS: In the present study, histological examination and in vitro cytotoxicity analysis according to DIN EN ISO 10993-5 were performed on a newly developed porcine vascular graft from a decellularized aorta. A conventional bovine graft was used as control. RESULTS: The ex vivo-histological analysis revealed the effectiveness of a new purification process on the microstructure and the removal of xenogeneic antigen-bearing structures in the new vessels. Furthermore, cell viability and cytotoxicity assays revealed full cytocompatibility. CONCLUSION: The novel graft shows no structural damage and gets completely decellularized by the purification process. Superior cytocompatibility, compared with the bovine-derived vascular graft, was demonstrated.

In Vivo Biocompatibility Analysis of a Novel Barrier Membrane Based on Bovine Dermis-Derived Collagen for Guided Bone Regeneration (GBR).

Collagen-based barrier membranes are nowadays the prevalent option for Guided Bone Regeneration (GBR) procedures. Xenogeneic collagen is highly biocompatible as it shares a similar structure to native human collagen, which prevents it from eliciting an exaggerated host immune response. Most commercially available collagen barrier membranes are porcine-derived, while bovine-derived alternatives are still rarely available. The aim of the present study was to investigate the tissue responses and the barrier functionality of a novel GBR membrane composed of bovine collagen type I (BM). Therefore, the subcutaneous implantation model in Wistar rats was performed to compare the novel medical device with two already clinically used native porcine-based barrier membranes, i.e., Jason® membrane (JM) and Bio-Gide® (BG), at 10-, 30-, 60-, and 90-days post implantationem. Histochemical and immunohistochemical stains were used for histopathological evaluation including a biocompatibility scoring according to the DIN EN ISO 10993-6 norm as well as histomorphometrical analyses of the occurrence of M1 and M2 macrophages and the transmembraneous vascularization. The bovine membrane exhibited a host tissue reaction that was comparable to both control materials, which was verified by the scoring results and the histomorphometrical macrophage measurements. Moreover, the novel membrane exhibited an integration pattern without material fragmentation up to day 60. At day 90, material fragmentation was observable that allowed for "secondary porosity" including transmembrane vascularization. The results of this study suggest that the novel bovine barrier membrane is fully biocompatible and suitable for indications that require GBR as a suitable alternative to porcine-sourced barrier membranes.

Comparison of the Validity of Enzymatic and Immunohistochemical Detection of Tartrate-resistant Acid Phosphatase (TRAP) in the Context of Biocompatibility Analyses of Bone Substitutes.

BACKGROUND/AIM: Macrophages and biomaterial-induced multinucleated giant cells (BMGCs) are central elements in the tissue reaction cascade towards bone substitute materials (BSM). The enzymatic detection of the lytic enzyme tartrate-resistant acid phosphatase (TRAP) has manifoldly been used to examine the so-called "bioactivity" of BSM. The present study aimed to compare the detection validity and expression pattern of the TRAP enzyme using enzymatic and immunohistochemical detection methods in the context of biocompatibility analyses of BSM. PATIENTS AND METHODS: Biopsies from 8 patients were analyzed after sinus augmentation with a xenogeneic bone substitute. Analysis of both macrophage and BMGC polarization were performed by histochemical TRAP detection and immunohistochemical detection of TRAP5a. Histomorphometrical analysis was used for comparison of the TRAP detection of BMGCs. RESULTS: The enzymatic TRAP detection method revealed that in 7 out of 8 biopsies only single cells were TRAP-positive, whereas most of the cells and especially the BMGCs were TRAP-negative. The immunohistochemical detection of TRAP5a showed moderate numbers of stained mononuclear cells, while the majority of the BMGCs showed signs of TRAP5a-expression. The enzymatic TRAP detection was comparable to the results obtained via immunohistochemistry only in one case. The histomorphometrical analysis showed that significantly more mononuclear and multinucleated TRAP-positive cells were found using immunohistochemical TRAP5a-staining compared to the enzymatic TRAP detection method. Also, significantly more TRAP-negative BMGCs were found using the enzymatic TRAP detection. CONCLUSION: The immunohistochemical detection of TRAP is more accurate for examination of the bioactivity and cellular degradability of BSM.

In Vivo Analysis of the Immune Response to Strontium- and Copper-doped Bioglass.

BACKGROUND: Bioglass is a highly adoptable bone substitute material which can be combined with so-called therapeutic ions. However, knowledge is poor regarding the influence of therapeutic ions on immune reactions and associated bone healing. Thus, the aim of this work was to investigate the influence of strontium- and copper-doped bioglass on the induction of M1 and M2 macrophages, as well as vascularization. MATERIALS AND METHODS: Two types of alkali glass were produced based on ICIE16 bioglass via the melt-quench method with the addition of 5 wt% copper or strontium (ICIE16-Cu and ICIE16-Sr). Pure ICIE16 and 45S5 bioglass were used as control materials. The ion release and chemical composition of the bioglass were investigated, and an in vivo experiment was subcutaneously performed on Sprague-Dawley rats. RESULTS: Scanning electron microscopy revealed significant differences in the surface morphology of the bioglass materials. Energy dispersive X-ray spectroscopy confirmed the efficiency of the doping process by showing the ion-release kinetics. ICIE16-Cu exhibited a higher ion release than ICIE16-Sr. ICIE16-Cu induced low immune cell migration and triggered not only a low number of M1 and M2 macrophages but also of blood vessels. ICIE16-Sr induced higher numbers of M1 macrophages after 30 days. Both bioglass types induced numbers of M2 macrophages comparable with those found in the control groups. CONCLUSION: Bioglass doping with copper and strontium did not significantly influence the foreign body response nor vascularization of the implantation bed in vivo. However, all the studied bioglass materials seemed to be biocompatible.

In Vitro Investigation of Jellyfish Collagen as a Tool in Cell Culture and (Bone) Tissue Engineering.

BACKGROUND/AIM: Jellyfish collagen serves as a competitive alternative to mammalian-sourced collagen in many practical aspects. For instance, jellyfish collagen lacks religious constraints when compared to bovine or porcine sources and promises batch-to-batch consistency. Another advantage is its structural similarity with many mammalian collagen types, providing a biocompatible matrix for different cell types as "collagen type 0". This paper intends to investigate jellyfish collagen (Jellagen®) in two applications. This investigation aims to establish an initial understanding of jellyfish collagen in biotechnology. More specifically, in cell culture and the field of tissue engineering. MATERIALS AND METHODS: The jellyfish collagen was comparatively tested as a coating material for multi-well plates as one of the most extensively used tools in cell culture and in the form of three-dimensional (3D) scaffolds intended for bone tissue engineering (BTE) applications. Both, the coated well plates and the scaffolds were seeded with fibroblasts and pre-osteoblasts, separately. In vitro cytocompatibility assays in accordance with EN ISO 10993-5/-12 regulations and LIVE-DEAD-stainings were carried out to study the cell viability, cytotoxicity and proliferation of these two cell lines. RESULTS: The results showed that collagen extracted from R. pulmo jellyfish can be an alternative to mammalian-derived collagen. Fibroblasts showed comparable cell viability to the medium control and an increased cell proliferation on the well plates indicating that these coated well plates can be used in cell culture, particularly in biocompatibility studies of biomaterials (as fibroblasts are used in this respective field extensively). The viability of pre-osteoblasts significantly exceeded the medium control in case of the jellyfish 3D scaffolds. CONCLUSION: These cells exhibited favorable healthy behavior on this marine collagen, suggesting that Jellagen® collagen can be used in studies of (bone) tissue regeneration and especially as scaffolds in BTE. In conclusion, jellyfish collagen provides biocompatibility and adhesive properties for both cell culture and BTE applications.

Comparative In Vivo Analysis of the Integration Behavior and Immune Response of Collagen-Based Dental Barrier Membranes for Guided Bone Regeneration (GBR).

Collagen-based resorbable barrier membranes have been increasingly utilized for Guided Bone Regeneration (GBR), as an alternative to non-resorbable synthetic membranes that require a second surgical intervention for removal. One of the most important characteristics of a resorbable barrier membrane is its mechanical integrity that is required for space maintenance and its tissue integration that plays a crucial role in wound healing and bone augmentation. This study compares a commercially available porcine-derived sugar-crosslinked collagen membrane with two non-crosslinked collagen barrier membranes. The material analysis provides an insight into the influence of manufacturing on the microstructure. In vivo subcutaneous implantation model provides further information on the host tissue reaction of the barrier membranes, as well as their tissue integration patterns that involve cellular infiltration, vascularization, and degradation. The obtained histochemical and immunohistochemical results over three time points (10, 30, and 60 days) showed that the tissue response to the sugar crosslinked collagen membrane involves inflammatory macrophages in a comparable manner to the macrophages observed in the surrounding tissue of the control collagen-based membranes, which were proven as biocompatible. The tissue reactions to the barrier membranes were additionally compared to wounds from a sham operation. Results suggest wound healing properties of all the investigated barrier membranes. However, the sugar-crosslinked membrane lacked in cellular infiltration and transmembraneous vascularization, providing an exclusive barrier function in GBR. Moreover, this membrane maintained a similar swelling ratio over examined timepoints, which suggests a very slow degradation pattern and supports its barrier function. Based on the study results, which showed biocompatibility of the sugar crosslinked membrane and its stability up to 60 days post-implantation, it can be concluded that this membrane may be suitable for application in GBR as a biomaterial with exclusive barrier functionality, similar to non-resorbable options.

The Granule Size Mediates the In Vivo Foreign Body Response and the Integration Behavior of Bone Substitutes.

The physicochemical properties of synthetically produced bone substitute materials (BSM) have a major impact on biocompatibility. This affects bony tissue integration, osteoconduction, as well as the degradation pattern and the correlated inflammatory tissue responses including macrophages and multinucleated giant cells (MNGCs). Thus, influencing factors such as size, special surface morphologies, porosity, and interconnectivity have been the subject of extensive research. In the present publication, the influence of the granule size of three identically manufactured bone substitute granules based on the technology of hydroxyapatite (HA)-forming calcium phosphate cements were investigated, which includes the inflammatory response in the surrounding tissue and especially the induction of MNGCs (as a parameter of the material degradation). For the in vivo study, granules of three different size ranges (small = 0.355-0.5 mm; medium = 0.5-1 mm; big = 1-2 mm) were implanted in the subcutaneous connective tissue of 45 male BALB/c mice. At 10, 30, and 60 days post implantationem, the materials were explanted and histologically processed. The defect areas were initially examined histopathologically. Furthermore, pro- and anti-inflammatory macrophages were quantified histomorphometrically after their immunohistochemical detection. The number of MNGCs was quantified as well using a histomorphometrical approach. The results showed a granule size-dependent integration behavior. The surrounding granulation tissue has passivated in the groups of the two bigger granules at 60 days post implantationem including a fibrotic encapsulation, while a granulation tissue was still present in the group of the small granules indicating an ongoing cell-based degradation process. The histomorphometrical analysis showed that the number of proinflammatory macrophages was significantly increased in the small granules at 60 days post implantationem. Similarly, a significant increase of MNGCs was detected in this group at 30 and 60 days post implantationem. Based on these data, it can be concluded that the integration and/or degradation behavior of synthetic bone substitutes can be influenced by granule size.

The Early Fragmentation of a Bovine Dermis-Derived Collagen Barrier Membrane Contributes to Transmembraneous Vascularization-A Possible Paradigm Shift for Guided Bone Regeneration.

Collagen-based barrier membranes are an essential component in Guided Bone Regeneration (GBR) procedures. They act as cell-occlusive devices that should maintain a micromilieu where bone tissue can grow, which in turn provides a stable bed for prosthetic implantation. However, the standing time of collagen membranes has been a challenging area, as native membranes are often prematurely resorbed. Therefore, consolidation techniques, such as chemical cross-linking, have been used to enhance the structural integrity of the membranes, and by consequence, their standing time. However, these techniques have cytotoxic tendencies and can cause exaggerated inflammation and in turn, premature resorption, and material failures. However, tissues from different extraction sites and animals are variably cross-linked. For the present in vivo study, a new collagen membrane based on bovine dermis was extracted and compared to a commercially available porcine-sourced collagen membrane extracted from the pericardium. The membranes were implanted in Wistar rats for up to 60 days. The analyses included well-established histopathological and histomorphometrical methods, including histochemical and immunohistochemical staining procedures, to detect M1- and M2-macrophages as well as blood vessels. Initially, the results showed that both membranes remained intact up to day 30, while the bovine membrane was fragmented at day 60 with granulation tissue infiltrating the implantation beds. In contrast, the porcine membrane remained stable without signs of material-dependent inflammatory processes. Therefore, the bovine membrane showed a special integration pattern as the fragments were found to be overlapping, providing secondary porosity in combination with a transmembraneous vascularization. Altogether, the bovine membrane showed comparable results to the porcine control group in terms of biocompatibility and standing time. Moreover, blood vessels were found within the bovine membranes, which can potentially serve as an additional functionality of barrier membranes that conventional barrier membranes do not provide.

3D Printing for Soft Tissue Regeneration and Applications in Medicine.

The use of additive manufacturing (AM) technologies is a relatively young research area in modern medicine. This technology offers a fast and effective way of producing implants, tissues, or entire organs individually adapted to the needs of a patient. Today, a large number of different 3D printing technologies with individual application areas are available. This review is intended to provide a general overview of these various printing technologies and their function for medical use. For this purpose, the design and functionality of the different applications are presented and their individual strengths and weaknesses are explained. Where possible, previous studies using the respective technologies in the field of tissue engineering are briefly summarized.