A feature of maternal sleep apnea during gestation causes autism-relevant neuronal and behavioral phenotypes in offspring.

Mounting epidemiologic and scientific evidence indicates that many psychiatric disorders originate from a complex interplay between genetics and early life experiences, particularly in the womb. Despite decades of research, our understanding of the precise prenatal and perinatal experiences that increase susceptibility to neurodevelopmental disorders remains incomplete. Sleep apnea (SA) is increasingly common during pregnancy and is characterized by recurrent partial or complete cessations in breathing during sleep. SA causes pathological drops in blood oxygen levels (intermittent hypoxia, IH), often hundreds of times each night. Although SA is known to cause adverse pregnancy and neonatal outcomes, the long-term consequences of maternal SA during pregnancy on brain-based behavioral outcomes and associated neuronal functioning in the offspring remain unknown. We developed a rat model of maternal SA during pregnancy by exposing dams to IH, a hallmark feature of SA, during gestational days 10 to 21 and investigated the consequences on the offspring's forebrain synaptic structure, synaptic function, and behavioral phenotypes across multiples stages of development. Our findings represent a rare example of prenatal factors causing sexually dimorphic behavioral phenotypes associated with excessive (rather than reduced) synapse numbers and implicate hyperactivity of the mammalian target of rapamycin (mTOR) pathway in contributing to the behavioral aberrations. These findings have implications for neuropsychiatric disorders typified by superfluous synapse maintenance that are believed to result, at least in part, from largely unknown insults to the maternal environment.

Bone regeneration capacity of newly developed spherical magnesium phosphate cement granules.

OBJECTIVES: Magnesium phosphate-based cements begin to catch more attention as bone substitute materials and especially as alternatives for the more commonly used calcium phosphates. In bone substitutes for augmentation purposes, atraumatic materials with good biocompatibility and resorbability are favorable. In the current study, we describe the in vivo testing of novel bone augmentation materials in form of spherical granules based on a calcium-doped magnesium phosphate (CaMgP) cement. MATERIALS AND METHODS: Granules with diameters between 500 and 710 µm were fabricated via the emulsification of CaMgP cement pastes in a lipophilic liquid. As basic material, two different CaMgP formulations were used. The obtained granules were implanted into drill hole defects at the distal femoral condyle of 27 New Zealand white rabbits for 6 and 12 weeks. After explantation, the femora were examined via X-ray diffraction analysis, histological staining, radiological examination, and EDX measurement. RESULTS: Both granule types display excellent biocompatibility without any signs of inflammation and allow for proper bone healing without the interposition of connective tissue. CaMgP granules show a fast and continuous degradation and enable fully adequate bone regeneration. CONCLUSIONS: Due to their biocompatibility, their degradation behavior, and their completely spherical morphology, these CaMgP granules present a promising bone substitute material for bone augmentation procedures, especially in sensitive areas. CLINICAL RELEVANCE: The mostly insufficient local bone supply after tooth extractions complicates prosthetic dental restoration or makes it even impossible. Therefore, bone augmentation procedures are oftentimes inevitable. Spherical CaMgP granules may represent a valuable bone replacement material in many situations.

In vitro evaluation of antibacterial efficacy of vancomycin-loaded suture tapes and cerclage wires.

Usage of implants containing antibiotic agents has been a common strategy to prevent implant related infections in orthopedic surgery. Unfortunately, most implants with microbial repellent properties are characterized by accessibility limitations during daily clinical practice. Aim of this in vitro study was to investigate whether suture tapes and cerclage wires, which were treated with vancomycin, show a sustainable antibacterial activity. For this purpose, we used 24 stainless steel wire cerclages and 24 ultra-high molecular weight polyethylene and polyester suture tape test bodies. The test bodies were incubated for 30 min. in 100 mg/ml vancomycin solution or equivalent volumes of 0.9% NaCl. After measuring the initial solution uptake of the test bodies, antibacterial efficacy via agar diffusion test with Staphylococcus aureus and vancomycin elution tests were performed 1, 2, 3, and 6 days after incubation. Vancomycin-loaded tapes as well as vancomycin-loaded cerclage wires demonstrated increased bacterial growth inhibition when compared to NaCl-treated controls. Vancomycin-loaded tapes showed an additional twofold and eightfold increase of bacterial growth inhibition compared to vancomycin-loaded wires at day 1 and 2, respectively. Elution tests at day 1 revealed high levels of vancomycin concentration in vancomycin loaded tapes and wires. Additionally, the concentration in vancomycin loaded tapes was 14-fold higher when compared to vancomycin loaded wires. Incubating suture tapes and cerclage wires in vancomycin solution showed a good short-term antibacterial activity compared to controls. Considering the ease of vancomycin application on suture tapes or wires, our method could represent an attractive therapeutic strategy in biofilm prevention in orthopedic surgery.

Critical parameters in cultivation of experimental biofilms using the example of Pseudomonas fluorescens.

Formation and treatment of biofilms present a great challenge for health care and industry. About 80% of human infections are associated with biofilms including biomaterial centered infections, like infections of prosthetic heart valves, central venous catheters, or urinary catheters. Additionally, biofilms can cause food and drinking water contamination. Biofilm research focusses on application of experimental biofilm models to study initial adherence processes, to optimize physico-chemical properties of medical materials for reducing interactions between materials and bacteria, and to investigate biofilm treatment under controlled conditions. Exploring new antimicrobial strategies plays a key role in a variety of scientific disciplines, like medical material research, anti-infectious research, plant engineering, or wastewater treatment. Although a variety of biofilm models exist, there is a lack of standardization for experimental protocols, and designing experimental setups remains a challenge. In this study, a number of experimental parameters critical for material research have been tested that influence formation and stability of an experimental biofilm using the non-pathogenic model strain of Pseudomonas fluorescens. These parameters include experimental time frame, nutrient supply, inoculum concentration, static and dynamic cultivation conditions, material properties, and sample treatment during staining for visualization of the biofilm. It was shown, that all tested parameters critically influence the experimental biofilm formation process. The results obtained in this study shall support material researchers in designing experimental biofilm setups.

In-Vitro Cytotoxicity Study: Cell Viability and Cell Morphology of Carbon Nanofibrous Scaffold/Hydroxyapatite Nanocomposites.

Electrospun carbon nanofibers (CNFs), which were modified with hydroxyapatite, were fabricated to be used as a substrate for bone cell proliferation. The CNFs were derived from electrospun polyacrylonitrile (PAN) nanofibers after two steps of heat treatment: stabilization and carbonization. Carbon nanofibrous (CNF)/hydroxyapatite (HA) nanocomposites were prepared by two different methods; one of them being modification during electrospinning (CNF-8HA) and the second method being hydrothermal modification after carbonization (CNF-8HA; hydrothermally) to be used as a platform for bone tissue engineering. The biological investigations were performed using in-vitro cell counting, WST cell viability and cell morphology after three and seven days. L929 mouse fibroblasts were found to be more viable on the hydrothermally-modified CNF scaffolds than on the unmodified CNF scaffolds. The biological characterizations of the synthesized CNF/HA nanofibrous composites indicated higher capability of bone regeneration.

Sex- and Region-Specific Differences in the Transcriptomes of Rat Microglia from the Brainstem and Cervical Spinal Cord.

The neural control system underlying breathing is sexually dimorphic with males being more vulnerable to dysfunction. Microglia also display sex differences, and their role in the architecture of brainstem respiratory rhythm circuitry and modulation of cervical spinal cord respiratory plasticity is becoming better appreciated. To further understand the molecular underpinnings of these sex differences, we performed RNA sequencing of immunomagnetically isolated microglia from brainstem and cervical spinal cord of adult male and female rats. We used various bioinformatics tools (Gene Ontology, Kyoto Encyclopedia of Genes and Genomes, Reactome, STRING, MAGICTRICKS) to functionally categorize identified gene sets, as well as to pinpoint common transcriptional gene drivers that may be responsible for the observed transcriptomic differences. We found few sex differences in the microglial transcriptomes derived from the brainstem, but several hundred genes were differentially expressed by sex in cervical spinal microglia. Comparing brainstem and spinal microglia within and between sexes, we found that the major factor guiding transcriptomic differences was central nervous system (CNS) location rather than sex. We further identified key transcriptional drivers that may be responsible for the transcriptomic differences observed between sexes and CNS regions; enhancer of zeste homolog 2 emerged as the predominant driver of the differentially downregulated genes. We suggest that functional gene alterations identified in metabolism, transcription, and intercellular communication underlie critical microglial heterogeneity and sex differences in CNS regions that contribute to respiratory disorders categorized by dysfunction in neural control. These data will also serve as an important resource data base to advance our understanding of innate immune cell contributions to sex differences and the field of respiratory neural control. SIGNIFICANCE STATEMENT: The contributions of central nervous system (CNS) innate immune cells to sexually dimorphic differences in the neural circuitry controlling breathing are poorly understood. We identify key transcriptomic differences, and their transcriptional drivers, in microglia derived from the brainstem and the C3-C6 cervical spinal cord of healthy adult male and female rats. Gene alterations identified in metabolism, gene transcription, and intercellular communication likely underlie critical microglial heterogeneity and sex differences in these key CNS regions that contribute to the neural control of breathing.

Biofabrication of cell-loaded 3D spider silk constructs.

Biofabrication is an emerging and rapidly expanding field of research in which additive manufacturing techniques in combination with cell printing are exploited to generate hierarchical tissue-like structures. Materials that combine printability with cytocompatibility, so called bioinks, are currently the biggest bottleneck. Since recombinant spider silk proteins are non-immunogenic, cytocompatible, and exhibit physical crosslinking, their potential as a new bioink system was evaluated. Cell-loaded spider silk constructs can be printed by robotic dispensing without the need for crosslinking additives or thickeners for mechanical stabilization. Cells are able to adhere and proliferate with good viability over at least one week in such spider silk scaffolds. Introduction of a cell-binding motif to the spider silk protein further enables fine-tuned control over cell-material interactions. Spider silk hydrogels are thus a highly attractive novel bioink for biofabrication.

S. aureus develops biofilms on titanium within 2 weeks in vitro.

BACKGROUND: The formation of biofilms, characterized by cell aggregation and extracellular polymeric substance (EPS) production, is a common feature of periprosthetic joint infections (PJI). OBJECTIVE: The current study aimed to investigate the development of biofilm features in vitro within less than 3 weeks by Staphylococcus aureus isolated from PJIs. METHODS: Biofilms were grown on sandblasted titanium discs, and fluorescence spectroscopy and microscopy were used to observe biofilm maturation for 21 days. RESULTS: DNA mass decreased initially, then increased from day 5 onwards, and decreased again after day 7. The proportion of living to dead bacteria oscillated until day 7 and increased at day 10 for strain A and day 14 for strain B. EPS mass decreased initially and then continuously increased. Multilayer bacterial organization was observed at day 7. CONCLUSION: Cell aggregation occurred during the first week, followed by EPS production in the second week, and characteristic biofilm features were observed within 1 to 2 weeks.

Degradation and Bone-Contact Biocompatibility of Two Drillable Magnesium Phosphate Bone Cements in an In Vivo Rabbit Bone Defect Model.

The use of bone-cement-enforced osteosynthesis is a growing topic in trauma surgery. In this context, drillability is a desirable feature for cements that can improve fracture stability, which most of the available cement systems lack. Therefore, in this study, we evaluated a resorbable and drillable magnesium-phosphate (MgP)-based cement paste considering degradation behavior and biocompatibility in vivo. Two different magnesium-phosphate-based cement (MPC) pastes with different amounts of phytic acid (IP 6) as setting retarder (MPC 22.5 and MPC 25) were implanted in an orthotopic defect model of the lateral femoral condyle of New Zealand white rabbits for 6 weeks. After explantation, their resorption behavior and material characteristics were evaluated by means of X-ray diffraction (XRD), porosimetry measurement, histological staining, peripheral quantitative computed tomography (pQCT), cone-beam computed tomography (CBCT) and biomechanical load-to-failure tests. Both cement pastes displayed comparable results in mechanical strength and resorption kinetics. Bone-contact biocompatibility was excellent without any signs of inflammation. Initial resorption and bone remodeling could be observed. MPC pastes with IP 6 as setting retardant have the potential to be a valuable alternative in distinct fracture patterns. Drillability, promising resorption potential and high mechanical strength confirm their suitability for use in clinical routine.

Corrigendum to "Degradation of 3D-printed magnesium phosphate ceramics in vitro and a prognosis on their bone regeneration potential" [Bioact. Mater. 19 (2023) 376-391/22].

[This corrects the article DOI: 10.1016/j.bioactmat.2022.04.015.].

Biomimetic Connection of Transcutaneous Implants with Skin.

Bacterial infection is a crucial complication in implant restoration, in particular in permanent skin-penetrating implants. Therein, the resulting gap between transcutaneous implant and skin represents a permanent infection risk, limiting the field of application and the duration of application. To overcome this limitation, a tight physiological connection is required to achieve a biological and mechanical welding for a long-term stable closure including self-healing probabilities. This study describes a new approach, wherein the implant is connected covalently to a highly porous electrospun fleece featuring physiological dermal integration potential. The integrative potential of the scaffold is shown in vitro and confirmed in vivo, further demonstrating tissue integration by neovascularization, extracellular matrix formation, and prevention of encapsulation. To achieve a covalent connection between fleece and implant surface, self-initiated photografting and photopolymerization of hydroxyethylmethacrylate is combined with a new crosslinker (methacrylic acid coordinated titanium-oxo clusters) on proton-abstractable implant surfaces. For implant modification, the attached fleece is directed perpendicular from the implant surface into the surrounding dermal tissue. First in vitro skin implantations demonstrate the implants' dermal integration capability as well as wound closure potential on top of the fleece by epithelialization, establishing a bacteria-proof and self-healing connection of skin and transcutaneous implant.

Formation and properties of magnesium-ammonium-phosphate hexahydrate biocements in the Ca-Mg-PO4 system.

Calcium substituted trimagnesium phosphate with the general formula Ca(x)Mg((3-x))(PO(4))(2) (0 < x < 1.5) was synthesized by calcination of powder mixtures with the appropriate stoichiometry and reacted with 3.5 M diammonium hydrogenphosphate solution to form a cementitious matrix of magnesium ammonium phosphate hexahydrate (struvite). The degree of ionic substitution was shown to influence physical cement properties; clinically suitable cement formulations with setting times in the range 5-15 min and compressive strengths of >50 MPa were obtained for x ≤ 0.75 together with a grinding time ≥ 1 h and a powder to liquid ratio ≥ 2.5 g/ml. The cement cytocompatibility was investigated by culturing human osteoblast cell line MG63 on cement surfaces demonstrating pronounced cell growth during 13 days cultivation.

Hard implant coatings with antimicrobial properties.

Infection of orthopaedic implants often leads to inflammation immediately after surgery and increases patient morbidity due to repetitive operations. Silver ions have been shown to combine good biocompatibility with a low risk of inducing bacterial resistance. In this study a physical vapour deposition system using both arc deposition and magnetron sputtering has been utilized to produce silver ion doped TiN coatings on Ti substrates. This biphasic system combines the advantages of silver induced bactericidity with the good mechanical properties of TiN. Crystallographic analysis by X-ray diffraction showed that silver was deposited as well in its elementary form as it was incorporated into the crystal lattice of TiN, which resulted in increasing hardness of the TiN-coatings. Elution experiments revealed a continuous release of Ag ions in phosphate buffered saline. The coatings showed significant inhibitory effects on the growth of Staphylococcus epidermidis and Staphylococcus aureus and practically no cell-toxicity in cytocompatibility tests.

Alzheimer's Disease, Sleep Disordered Breathing, and Microglia: Puzzling out a Common Link.

Sleep Disordered Breathing (SDB) and Alzheimer's Disease (AD) are strongly associated clinically, but it is unknown if they are mechanistically associated. Here, we review data covering both the cellular and molecular responses in SDB and AD with an emphasis on the overlapping neuroimmune responses in both diseases. We extensively discuss the use of animal models of both diseases and their relative utilities in modeling human disease. Data presented here from mice exposed to intermittent hypoxia indicate that microglia become more activated following exposure to hypoxia. This also supports the idea that intermittent hypoxia can activate the neuroimmune system in a manner like that seen in AD. Finally, we highlight similarities in the cellular and neuroimmune responses between SDB and AD and propose that these similarities may lead to a pathological synergy between SDB and AD.

Influence of Tranexamic Acid on Elution Characteristics and Compressive Strength of Antibiotic-Loaded PMMA-Bone Cement with Gentamicin.

PURPOSE: The topical application of tranexamic acid (TXA) into the joint space during total joint arthroplasty (TJA) with no increase of complications, has been widely reported. We investigated the influence of TXA on antibiotic release, activity of the released antibiotic against a clinical isolate of S. aureus, and compressive strength of a widely used commercially prepared gentamicin-loaded cement brand (PALACOS R + G). METHOD: 12 bone cement cylinders (diameter and height = 6 and 12 mm, respectively) were molded. After curing in air for at least 1 h, six of the cylinders were completely immersed in 5 mL of fetal calf serum (FCS) and the other six were completely immersed in a solution consisting of 4.9 mL of FCS and 0.1 mL (10 mg) of TXA. Gentamicin elution tests were performed over 7 d. Four hundred µL of the gentamicin eluate were taken every 24 h for the first 7 d without renewing the immersion fluid. The gentamicin concentration was determined in a clinical analyzer using a homogeny enzyme immuno-assay. The antimicrobial activity of the eluate, obtained after day 7, was tested. An agar diffusion test regime was used with Staphylococcus aureus. Bacteria were grown in a LB medium and plated on LB agar plates to get a bacterial lawn. Fifty µL of each eluate were pipetted on 12-mm diameter filter discs, which were placed in the middle of the agar gel. After 24 h of cultivation at 37 °C, the zone of inhibition (ZOI) for each specimen was measured. The compressive strength of the cements was determined per ISO 5833. RESULTS: At each time point in the gentamicin release test, the difference in gentamicin concentration, obtained from specimens immersed in the FCS solution only and those immersed in the FCS + TXA solution was not significant (p = 0.055-0.522). The same trend was seen in each of the following parameters, after 7 d of immersion: (1) Cumulative gentamicin concentration (p < 0.297); (2) gentamicin activity against S. aureus (strongly visible); (3) ZOI size (mostly > 20 mm) (p = 0.631); and (4) compressive strength (p = 0.262). CONCLUSIONS: For the PALACOS R + G specimens, the addition of TXA to FCS does not produce significant decreases in gentamicin concentration, in the activity of the gentamicin eluate against a clinical isolate of S. aureus, the zone of inhibition of S. aureus, and in the compressive strength of the cement, after 7 d of immersion in the test solution.

Osteoclast and osteoblast response to strontium-doped struvite coatings on titanium for improved bone integration.

To develop implants with improved bone ingrowth, titanium substrates were coated with homogeneous and dense struvite (MgNH4PO4·6H2O) layers by means of electrochemically assisted deposition. Strontium nitrate was added to the coating electrolyte in various concentrations, in order to fabricate Sr-doped struvite coatings with Sr loading ranging from 10.6 to 115 µg/cm2. It was expected and observed that osteoclast activity surrounding the implant was inhibited. The cytocompatibility of the coatings and the effect of Sr-ions in different concentrations on osteoclast formation were analyzed in vitro. Osteoclast differentiation was elucidated on morphological, biochemical as well as on gene expression level. It could be shown that moderate concentrations of Sr2+ had an inhibitory effect on osteoclast formation, while the growth of osteoblastic cells was not negatively influenced compared to pure struvite surfaces. In summary, the electrochemically deposited Sr-doped struvite coatings are a promising approach to improve bone implant ingrowth.

Prior Hypoxia Exposure Enhances Murine Microglial Inflammatory Gene Expression in vitro Without Concomitant H3K4me3 Enrichment.

Hypoxia (Hx) is a component of multiple disorders, including stroke and sleep-disordered breathing, which often precede or are comorbid with neurodegenerative diseases. However, little is known about how hypoxia affects the ability of microglia, resident CNS macrophages, to respond to subsequent inflammatory challenges that are often present during neurodegenerative processes. We, therefore, tested the hypothesis that hypoxia would enhance or "prime" microglial pro-inflammatory gene expression in response to a later inflammatory challenge without programmatically increasing basal levels of pro-inflammatory cytokine expression. To test this, we pre-exposed immortalized N9 and primary microglia to hypoxia (1% O2) for 16 h and then challenged them with pro-inflammatory lipopolysaccharide (LPS) either immediately or 3-6 days following hypoxic exposure. We used RNA sequencing coupled with chromatin immunoprecipitation sequencing to analyze primed microglial inflammatory gene expression and modifications to histone H3 lysine 4 trimethylation (H3K4me3) at the promoters of primed genes. We found that microglia exhibited enhanced responses to LPS 3 days and 6 days post-hypoxia. Surprisingly, however, the majority of primed genes were not enriched for H3K4me3 acutely following hypoxia exposure. Using the bioinformatics tool MAGICTRICKS and reversible pharmacological inhibition, we found that primed genes required the transcriptional activities of NF-κB. These findings provide evidence that hypoxia pre-exposure could lead to persistent and aberrant inflammatory responses in the context of CNS disorders.

Effect of strontium substitution on the material properties and osteogenic potential of 3D powder printed magnesium phosphate scaffolds.

3D powder printing is a versatile method for the fabrication of individual bone implants and was used for the processing of in vivo degradable ceramic scaffolds based on ammonium magnesium phosphate hexahydrate (struvite). In this study, synergetic effects could be achieved by the substitution of magnesium phosphate cements with strontium carbonate. This substitution resulted in 8.2 wt%, 16.4 wt%, and 24.6 wt% Sr2+ doped scaffolds, with a 1.9-3.1 times increased radiopacity compared to pure struvite. The maximal compressive strength of (16.1 ±â€¯1.1) MPa found for strontium substituted magnesium phosphate was in the range of cancelleous bone, which makes these 3D printed structures suitable for medical application in low-load-bearing bone areas. In an ion release study over a course of 18 days, the release of strontium, magnesium, calcium, and phosphate ions from scaffolds was analyzed by means of inductively coupled plasma mass spectrometry. Independent of the scaffold composition the Mg2+ concentrations (83-499 mg/l) continuously increased in the cell media. The Sr2+ release varied between 4.3 µg/day and 15.1 µg/day per g scaffold, corresponding to a Sr2+ concentration in media between 1.14 mg/l and 7.24 mg/l. Moreover, decreasing calcium and phosphate concentrations indicated the precipitation of an amorphous calcium phosphate phase. The superior osteogenic properties of strontium substituted magnesium phosphate, e.g. the increase of osteoblast activity and cell number and the simultaneous suppression of osteoclast differentiation could be verified in vitro by means of WST-assay, TRAP-staining, and SEM imaging.

Development and Bone Regeneration Capacity of Premixed Magnesium Phosphate Cement Pastes.

Magnesium phosphate cements (MPC) have been demonstrated to have a superior bone regeneration capacity due to their good solubility under in vivo conditions. While in the past only aqueous MPC pastes have been applied, the current study describes the fabrication and in vitro/in vivo testing of an oil-based calcium doped magnesium phosphate (CaMgP) cement paste. Premixed oil-based pastes with CaMgP chemistry combine the advantages of conventional MPC such as high mechanical strength and good resorbability with a prolonged shelf-life and an easier clinical handling. The pastes set in an aqueous environment and predominantly form struvite and achieve a compressive strength of ~8-10 MPa after setting. The implantation into a drill-hole defect at the distal femoral condyle of New Zealand white rabbits over a course of 6 and 12 weeks demonstrated good biocompatibility of the materials without the formation of soft connective tissue or any signs of inflammation. In contrast to a hydroxyapatite forming reference paste, the premixed CaMgP pastes showed subsequent degradation and bony regeneration. The CaMgP cement pastes presented herein are promising bone replacement materials with excellent material properties for an improved and facilitated clinical application.

Anisotropic Cryostructured Collagen Scaffolds for Efficient Delivery of RhBMP-2 and Enhanced Bone Regeneration.

In the treatment of bone non-unions, an alternative to bone autografts is the use of bone morphogenetic proteins (BMPs), e.g., BMP-2, BMP-7, with powerful osteoinductive and osteogenic properties. In clinical settings, these osteogenic factors are applied using absorbable collagen sponges for local controlled delivery. Major side effects of this strategy are derived from the supraphysiological doses of BMPs needed, which may induce ectopic bone formation, chronic inflammation, and excessive bone resorption. In order to increase the efficiency of the delivered BMPs, we designed cryostructured collagen scaffolds functionalized with hydroxyapatite, mimicking the structure of cortical bone (aligned porosity, anisotropic) or trabecular bone (random distributed porosity, isotropic). We hypothesize that an anisotropic structure would enhance the osteoconductive properties of the scaffolds by increasing the regenerative performance of the provided rhBMP-2. In vitro, both scaffolds presented similar mechanical properties, rhBMP-2 retention and delivery capacity, as well as scaffold degradation time. In vivo, anisotropic scaffolds demonstrated better bone regeneration capabilities in a rat femoral critical-size defect model by increasing the defect bridging. In conclusion, anisotropic cryostructured collagen scaffolds improve bone regeneration by increasing the efficiency of rhBMP-2 mediated bone healing.