Both preclinical in vivo experiments and clinicaltrials are indispensable for analysis of tissue reactions in evaluating the compatibility of biomaterials or medical devices, i.e. the cell types interacting with the material, integration or degradation behavior, implant bed vascularization and immunological response. In particular, both the histological workup (including the processes such as embedding, cutting, histochemical and immunohistochemical staining methods), as well as qualitative and quantitative analysis are crucial steps enabling the final evaluation of biocompatibility. We present a short overview of the most important steps of the different workup and analytical methods used in preclinical and clinical biopsies for both novice and experienced researchers in the field of biomaterial science.

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.

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.

Background/Aim: For the treatment of different tissue defects such as jawbone defects, open wound defect, chronic ulcers, dura mater defects and corneal defects, different biomaterials are available. The use of collagen-based materials for these applications has been significantly increased over the past decades due to its excellent biocompatibility and degradability. However, no transparent collagen-based biomaterial is available until now. Thus, a newly developed transparent collagen membrane (TCM) based on natural derived porcine pericardium, which offers numerous application possibilities, was developed. The present study aimed to analyze the in vitro and in vivo biocompatibility using established methods. Materials and Methods: The new TCM membrane and a commercially available collagen membrane (CM, Jason membrane, botiss biomaterials GmbH, Zossen, Germany) were tested for its in vitro cytocompatibility. Furthermore, the in vivo biocompatibility was analyzed using sham operations as control group. In vitro, cytocompatibility was tested in accordance with EN ISO 10993-5/-12 regulations and Live-Dead-stainings. In vivo, a subcutaneous implantation model in BALB/c mice was used and explants were prepared for analyses by established histological, immunohistochemical and histomorphometrical methods. Results: In vitro, both membranes showed promising cytocompatibility with a slightly better direct cell response in the Live-Dead staining assay for the TCM. In vivo, TCM induced a comparable inflammatory immune response after 10 and 30 days with comparable numbers of M1-and M2-macrophages as also found in the control group without biomaterial insertion. Conclusion: The newly transparent collagen membrane is fully biocompatible and is supporting safe clinical application in tissue repair and surgery.Tissue defects as a result of various causalities such as surgical interventions, infections or poorly healing wounds often require the use of regenerative materials. To this day, the use of autologous grafts is considered to be the gold standard (1-3). However, the removal of autologous tissue is associated with co-morbidities at the extraction site as well as a restrictive overall availability (1-3). Moreover, the success rates are dependent on the applied method, the donor with possible comorbidities (e.g. diabetes) as well as the skill of the surgeon (4).Medical devices based on collagen have manifoldly been identified as a reliable treatment option guiding wound healing processes due to the fact that collagen is the most abundant protein of the extracellular matrix guiding cellmatrix-interactions and creating a micro-environment for 2287 This article is freely accessible online.

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.