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.

In Vitro Cytocompatibility Analysis and Comparison of Different Hyaluronic Acid Fillers for Minimally Invasive Esthetics.

BACKGROUND/AIM: To overcome the natural visual consequences of the physiological aging process, the use of biodegradable fillers made of hyaluronic acid or sodium carboxymethyl cellulose is increasingly popular in modern esthetic medicine. Clinicians can choose from a wide range of fillers with variable compositions and rheological properties, and therefore with different application areas and injection depths. The aim of this study was to analyze and compare the most commonly used fillers for facial augmentation regarding their in vitro biocompatibility and to find potential correlations to their rheological properties. MATERIALS AND METHODS: In the present study, direct and indirect in vitro cytotoxicity analysis according to DIN EN ISO 10993-5 were performed on 39 different filler materials for facial augmentation. RESULTS: All fillers analyzed in this study overall showed satisfactory results in the direct and indirect cytocompatibility tests. While no material was outside the threshold values in the 2,3-bis-(2-methoxy-4-nitro-5-sulphenyl)-(2H)-tetrazolium-5-carboxanilide (XTT) cell viability and bromodeoxyuridine (BrdU) cell proliferation assays or in the live-dead staining, only 7 out of the 39 fillers reached the required values in the lactate dehydrogenase assay. CONCLUSION: All biodegradable fillers examined in this study were found to be sufficiently cytocompatible. Although the qualitative analysis of the test results showed differences between the fillers, no concrete correlation between test performance and composition or manufacturer of the fillers was found. Future efforts are required to provide clinicians with even better support in choosing the right filler for optimal outcome and patient satisfaction.

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.

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.

Republication: In Vitro and Ex Vivo Analysis of Collagen Foams for Soft and Hard Tissue Regeneration.

BACKGROUND/AIM: The aim of this study was the conception, production, material analysis and cytocompatibility analysis of a new collagen foam for medical applications. MATERIALS AND METHODS: After the innovative production of various collagen sponges from bovine sources, the foams were analyzed ex vivo in terms of their structure (including pore size) and in vitro in terms of cytocompatibility according to EN ISO 10993-5/-12. In vitro, the collagen foams were compared with the established biomaterials cerabone and Jason membrane. Materials cerabone and Jason membrane. RESULTS: Collagen foams with different compositions were successfully produced from bovine sources. Ex vivo, the foams showed a stable and long-lasting primary structure quality with a bubble area of 1,000 to 2,000 µm2 In vitro, all foams showed sufficient cytocompatibility. CONCLUSION: Collagen sponges represent a promising material for hard and soft tissue regeneration. Future studies could focus on integrating and investigating different additives in the foams.

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.

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 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.

Encapsulated vaterite-calcite CaCO3 particles loaded with Mg2+ and Cu2+ ions with sustained release promoting osteogenesis and angiogenesis.

Bioactive cations, including calcium, copper and magnesium, have shown the potential to become the alternative to protein growth factor-based therapeutics for bone healing. Ion substitutions are less costly, more stable, and more effective at low concentrations. Although they have been shown to be effective in providing bone grafts with more biological functions, the precise control of ion release kinetics is still a challenge. Moreover, the synergistic effect of three or more metal ions on bone regeneration has rarely been studied. In this study, vaterite-calcite CaCO3 particles were loaded with copper (Cu2+) and magnesium (Mg2+). The polyelectrolyte multilayer (PEM) was deposited on CaCuMg-CO3 particles via layer-by-layer technique to further improve the stability and biocompatibility of the particles and to enable controlled release of multiple metal ions. The PEM coated microcapsules were successfully combined with collagen at the outmost layer, providing a further stimulating microenvironment for bone regeneration. The in vitro release studies showed remarkably stable release of Cu2+ in 2 months without initial burst release. Mg2+ was released in relatively low concentration in the first 7 days. Cell culture studies showed that CaCuMg-PEM-Col microcapsules stimulated cell proliferation, extracellular maturation and mineralization more effectively than blank control and other microcapsules without collagen adsorption (Ca-PEM, CaCu-PEM, CaMg-PEM, CaCuMg-PEM). In addition, the CaCuMg-PEM-Col microcapsules showed positive effects on osteogenesis and angiogenesis in gene expression studies. The results indicate that such a functional and controllable delivery system of multiple bioactive ions might be a safer, simpler and more efficient alternative of protein growth factor-based therapeutics for bone regeneration. It also provides an effective method for functionalizing bone grafts for bone tissue engineering.

Lyophilized Human Bone Allograft as an Antibiotic Carrier: An In Vitro and In Vivo Study.

Background: Antibiotics delivered from implanted bone substitute materials (BSM) can potentially be used to prevent acute infections and biofilm formation, providing high concentrations of antibiotics at the surgical site without systemic toxicity. In addition, BSM should allow osteoconductivity supporting bone healing without further surgery. Promising results have been achieved using lyophilized bone allografts mixed with antibiotics. Methods: In this study specially prepared human bone allografts were evaluated as an antibiotic carrier in vitro and in vivo. The efficacy of different antibiotic-impregnated bone allografts was measured by drug release tests in vitro and in vivo and bacterial susceptibility tests using four bacterial species usually responsible for implant-associated infections. Results: The loading procedures of allograft bone substitutes with antibiotics were successful. Some of the antibiotic concentrations exceeded the MIC90 for up to 7 days in vitro and for up to 72 h in vivo. The susceptibility tests showed that S. epidermidis ATCC 12228 was the most susceptible bacterial species in comparison to the other strains tested for all antibiotic substances. Vancomycin and rifampicin showed the best results against standard and patient-isolated strains in vitro. In vivo, new bone formation was comparable in all study groups including the control group without antibiotic loading. Conclusions: Human bone allografts showed the capacity to act as customized loaded antibiotic carriers to prevent acute infections and should be considered in the management of bone infections in combination with systemic antimicrobial therapy.

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 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.

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 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 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.

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.

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.

Novel Histomorphometrical Approach to Evaluate the Integration Pattern and Functionality of Barrier Membranes.

GBR (guided bone regeneration) is a standard procedure for building up bony defects in the jaw. In this procedure, resorbable membranes made of bovine and porcine collagen are increasingly being used, which, in addition to many possible advantages, could have the potential disadvantage of a shorter barrier functionality, especially when augmenting large-volume defects. Thus, it is of importance to evaluate the integration behavior and especially the standing time of barrier membranes using specialized methods to predict its respective biocompatibility. This study is intended to establish a new histomorphometrical analysis method to quantify the integration rate of collagen-based barrier membranes. Three commercially available barrier membranes, i.e., non-crosslinked membranes (BioGide® and Jason® membrane), a ribose-crosslinked membrane (Ossix® Plus), and a newly developed collagen-hyaluronic acid-based (Coll-HA) barrier membrane were implanted in the subcutaneous tissue of 48 6-8-week-old Wistar rats. The explants, after three timepoints (10, 30, and 60 days), were processed and prepared into histological sections for histopathological (host tissue response) and histomorphometrical (cellular invasion) analyses. 10 days after implantation, fragmentation was not evident in any of the study groups. The sections of the Coll-HA, Jason® and BioGide® membranes showed a similar mild inflammatory reaction within the surrounding tissue and an initial superficial cell immigration. Only in the Ossix® Plus group very little inflammation and no cell invasion was detected. While the results of the three commercially available membranes remained intact in the further course of the study, only fragments of the Coll-HA membrane were found 30 and 60 days after implantation. Histomorphometrically, it can be described that although initially (at 10 days post-implantation) similar results were found in all study groups, after 30 days post-implantation the cellular penetration depth of the hyaluronic acid-collagen membrane was significantly increased with time (**** p < 0.0001). Similarly, the percentage of cellular invasion per membrane thickness was also significantly higher in the Coll-HA group at all timepoints, compared to the other membranes (**** p < 0.0001). Altogether, these results show that the histomorphometrical analysis of the cellular migration can act as an indicator of integration and duration of barrier functionality. Via this approach, it was possible to semi-quantify the different levels of cellular penetration of GBR membranes that were only qualitatively analyzed through histopathological approaches before. Additionally, the results of the histopathological and histomorphometrical analyses revealed that hyaluronic acid addition to collagen does not lead to a prolonged standing time, but an increased integration of a collagen-based biomaterial. Therefore, it can only partially be used in the dental field for indications that require fast resorbed membranes and a fast cell or tissue influx such as periodontal regeneration processes.