Pure Collagen Research Articles

Multivariate analysis of Raman spectra for discriminating human collagens: In vitro identification of extracellular matrix collagens produced by an osteosarcoma cell line

BackgroundThe NHS spends £4.3 billion annually to address musculoskeletal conditions, encompassing age-related bone disorders like osteoarthritis and osteoporosis. Traditional X-ray diagnostic methods are commonly employed for bone disorder diagnosis, primarily assessing gross anatomical bone structure changes. However, these methods are unable to identify subtle biochemical alterations within the bone. More detailed information, particularly about protein changes, may lead to enhanced diagnostics and treatment. Raman spectroscopy is a non-invasive, laser-based technique capable of detecting changes in the collagen component of bone. Despite its long-standing application in discerning mineral and protein changes within bone, there is limited evidence on Raman spectral signatures of purified human collagens and their differentiation. This study aimed to test the hypothesis that Raman spectroscopy could detect different types of collagen in the human body. ResultsA Raman microspectrometer with a 785 nm laser was used to measure unmineralized human collagens types I – VI and collagenous extracellular matrix (ECM) secreted by MG63 osteoblast-like cells. The results demonstrated the efficacy of Raman spectroscopy and subsequent multivariate analysis in distinguishing human collagen types I – VI. This implies that Raman spectroscopy, coupled with multivariate analysis, can identify pure human collagens and offers reference spectra similar to natural human collagen in the bone extracellular matrix. SignificanceThis study establishes Raman spectroscopy as a tool for identifying and characterizing human collagens, aiding in the diagnosis of connective tissue disorders. The creation of a spectral reference library for pure human collagen types I – VI holds potential for medical diagnostics, analytical chemistry, and materials science applications.

Elastic and recoverable sponges based on collagen/yeast β-glucan for quick hemostasis

In this investigation, we aimed to engineer sponges with exceptional mechanical and hemostatic capabilities for effective wound healing. By combining collagen, a stiff fibril protein in ECM, with β-glucan, an elastic and triple-helical polysaccharide from yeast cell wall, we prepared a series of composite sponges, designated as CY sponges. This material exhibited a uniform pore structure, displaying enhanced elasticity and shape recovery ability compared to pure collagen sponges. Also, the incorporation of Yeast β-glucan (YG) significantly improved the fluid absorption ability and stability of the sponges. In vitro hemostasis tests demonstrated that the CY sponges exhibited a notably lower in vitro coagulation index (19.21 %) compared to the collagen control (64.84 %), accompanied by superior erythrocyte (64.64 %) and platelet (64.95 %) adhesion properties. Animal studies further substantiated the sponge's hemostatic efficacy, as CY40 led to a reduction in average bleeding volume by 25.26 % and 28.97 %, and a shorter hemostatic time by 31.70 % and 30.77 % compared to collagen, indicating accelerated wound healing. These findings suggest that the addition of yeast β-glucan into collagen sponges can improve their elasticity, shape recovery ability, hemostatic performance and wound repair ability.

Enabling 3D bioprinting of cell-laden pure collagen scaffolds via tannic acid supporting bath.

The fabrication of cell-laden biomimetic scaffolds represents a pillar of tissue engineering and regenerative medicine (TERM) strategies, and collagen is the gold standard matrix for cells to be. In the recent years, extrusion 3D bioprinting introduced new possibilities to increase collagen scaffold performances thanks to the precision, reproducibility, and spatial control. However, the design of pure collagen bioinks represents a challenge, due to the low storage modulus and the long gelation time, which strongly impede the extrusion of a collagen filament and the retention of the desired shape post-printing. In this study, the tannic acid-mediated crosslinking of the outer layer of collagen is proposed as strategy to enable collagen filament extrusion. For this purpose, a tannic acid solution has been used as supporting bath to act exclusively as external crosslinker during the printing process, while allowing the pH- and temperature-driven formation of collagen fibers within the core. Collagen hydrogels (concentration 2-6mg/mL) were extruded in tannic acid solutions (concentration 5-20mg/mL). Results proved that external interaction of collagen with tannic acid during 3D printing enables filament extrusion without affecting the bulk properties of the scaffold. The temporary collagen-tannic acid interaction resulted in the formation of a membrane-like external layer that protected the core, where collagen could freely arrange in fibers. The precision of the printed shapes was affected by both tannic acid concentration and needle diameter and can thus be tuned. Altogether, results shown in this study proved that tannic acid bath enables collagen bioprinting, preserves collagen morphology, and allows the manufacture of a cell-laden pure collagen scaffold.

A high-strength collagen-based antimicrobial film grafted with ε-polylysine fabrication by riboflavin-mediated ultraviolet irradiation for pork preservation

In this study, a UV-cured collagen-based film (C-P-H film) with high mechanical strength and antimicrobial properties was developed by riboflavin-mediated ultraviolet irradiation of collagen solution containing histidine-modified ε-polylysine. Fourier transform infrared analysis indicated that covalent cross-linking was formed between the collagen molecule and the histidine-grafted ε-polylysine. Compared with the pure collagen film, the C-P-H film containing 5 wt% histidine-modified ε-polylysine showed higher tensile strength (145.98 MPa), higher thermal denaturation temperature (76.5 °C), lower water vapor permeability (5.54 × 10−11 g m−1 s−1 Pa) and excellent antimicrobial activities against Escherichia coli and Staphylococcus aureus. In addition, the wrapping of the C-P-H film effectively inhibited bacterial growth of pork during storage time, successfully prolonging the shelf-life of pork by approximately 4 days compared to that of plastic wrap. These results suggested that collagen-based film grafted with histidine-modified ε-polylysine via riboflavin-mediated ultraviolet irradiation process had a great potential for pork preservation.

Collagen as Bioink for Bioprinting: A Comprehensive Review.

Biomaterials made using collagen are successfully used as a three-dimensional (3D) substrate for cell culture and considered to be promising scaffolds for creating artificial tissues. An important task that arises for engineering such materials is the simulation of physical and morphological properties of tissues, which must be restored or replaced. Modern additive technologies, including 3D bioprinting, can be applied to successfully solve this task. This review provides the latest evidence on advances of 3D bioprinting with collagen in the field of tissue engineering. It contains modern approaches for printing pure collagen bioinks consisting only of collagen and cells, as well as the obtained results from the use of pure collagen bioinks in different fields of tissue engineering.

Effects of pre-treatment, historical age, and sample characteristics on the stable isotope analyses of killer whale (Orcinus orca) bone.

Stable isotope analysis of bone provides insight into animal foraging and allows for ecological reconstructions over time, however pre-treatment is required to isolate collagen. Pre-treatments typically consist of demineralization to remove inorganic components and/or lipid extraction to remove fats, but these protocols can differentially affect stable carbon (δ13C) and nitrogen (δ15N) isotope values depending on the chemicals, tissues, and/or species involved. Species-specific methodologies create a standard for comparability across studies and enhance understanding of collagen isolation from modern cetacean bone. Elemental analyzers coupled to isotope ratio mass spectrometers were used to measure the δ13C and δ15N values of powdered killer whale (Orcinus orca) bone that was intact (control) or subjected to one of three experimental conditions: demineralized, lipid-extracted, and both demineralized and lipid-extracted. Additionally, C:N ratios were evaluated as a proxy for collagen purity. Lastly, correlations were examined between control C:N ratios vs. historical age and control C:N ratios vs. sample characteristics. No significant differences in the δ15N values were observed for any of the experimental protocols. However, the δ13C values were significantly increased by all three experimental protocols: demineralization, lipid extraction, and both treatments combined. The most influential protocol was both demineralization and lipid extraction. Measures of the C:N ratios were also significantly lowered by demineralization and both treatments combined, indicating the material was closer to pure collagen after the treatments. Collagen purity as indicated via C:N ratio was not correlated with historical age nor sample characteristics. If only the δ15N values from killer whale bone are of interest for analysis, no pre-treatment seems necessary. If the δ13C values are of interest, samples should be both demineralized and lipid-extracted. As historical age and specimen characteristics are not correlated with sample contamination, all samples can be treated equally.

Performance of a multiphase bioactive socket plug with a barrier function for alveolar ridge preservation

The natural healing process of extraction socket and traditional socket plug material could not prevent buccal bone wall resorption and down growth of epithelium from the socket orifice. A multiphase bioactive socket plug (BP) is designed to overcome the natural healing process by maintaining the three-dimensional (3D) volume of extraction sockets, particularly in sockets with wall defects, and later provide sufficient alveolar bone volume for implant placement. The study aimed to fabricate and evaluate the physical, chemical, and biological performance of BP in vitro. The BP was fabricated through freeze-drying and layer-by-layer assembly, comprised of a base serving as a scaffold, a central portion for promoting bone regeneration, an upper buccal portion for maintaining alveolar socket dimension with a covering collagen membrane (Memb) on the top and upper buccal surface to prevent soft tissue infiltration. The BP as the experimental group and a pure collagen plug (CP) as the control group were investigated and compared. Radiograph, scanning electron microscopy, and energy-dispersive spectroscopy mapping confirmed that the four-part BP was successfully assembled and fabricated. Swelling rate analysis indicated that BP, CP, and Memb reached swelling equilibrium within 1 hour. BP exhibited a high remaining weight percentage in collagenase solution (68.81 ± 2.21% on day 90) and sustained calcium ion release, reaching the maximum 0.13 ± 0.04 mmol l−1 on day 14. In biological assays, BP exhibited excellent cell proliferation (The OD value increased from 0.02 on day 1 to 0.23 on day 21.). The BP group exhibited higher alkaline phosphatase activity and osteocalcin content than the CP group within 21 days. Memb and BP exhibited outstanding barrier function, as evidenced by Hematoxylin and eosin staining. In summary, the multiphase bioactive socket plug represents a promising scaffold for alveolar ridge preservation application.

Zirconium ion mediated collagen nanofibrous hydrogels with high mechanical strength

Low mechanical strength is still the key question for collagen hydrogel consisting of nanofibrils as hard tissue repair scaffolds with no loss of biological function. In this work, novel collagen nanofibrous hydrogels with high mechanical strength were fabricated based on the pre-protection of trisodium citrate masked Zr(SO4)2 solution for collagen self-assembling nanofibrils and then further coordination with Zr(SO4)2 solution. The mature collagen nanofibrils with d-period were observed in Zr(IV) mediated collagen hydrogels by AFM when the Zr(IV) concentration was ≥ 10 mmol/L, and the distribution of zirconium element was uniform. Due to the coordination of Zr(IV) with ─COOH, ─NH2 and ─OH within collagen and the tighter entanglement of collagen nanofibrils, the elastic modulus and compressive strength of Zr(IV) mediated collagen nanofibrous hydrogel were 208.3 and 1103.0 kPa, which were approximate 77 and 12 times larger than those of pure collagen hydrogel, respectively. Moreover, the environmental stability such as thermostability, swelling ability and biodegradability got outstanding improvements and could be regulated by Zr(IV) concentration. Most importantly, the resultant hydrogel showed excellent biocompatibility and even accelerated cell proliferation.

Harnessing oriented arrangement of collagen fibers by 3D printing for enhancing mechanical and osteogenic properties of mineralized collagen scaffolds

As the structural basis of connective and load-bearing tissues, collagen fibers with orientation play an important role in the mechanical properties and physiological and biochemical functions of the tissues, but viable methods for preparing scaffolds with highly oriented collagenous structure still need to be further studied. In this study, pure collagen was used as printing ink to 3D printing. Harnessing oriented collagen fiber structure by 3D printing for promoting mechanical and osteogenic properties of scaffolds. The scaffolds with different printed angles and thicknesses were prepared to fit the bone defect site and realize personalized customization. The orientation assembly of collagen fibers was promoted by shear force action of 3D printing, the regular arrangement of collagen fibers and stabilization of fiber structure were promoted by pH adjustment and glutaraldehyde cross-linking, and the collagen fibers were mineralized by cyclic mineralization method. The microscopic morphology of fiber arrangement in the scaffolds were investigated by scanning electron microscopy. Results demonstrated that collagen fibers were changed from non-oriented to oriented after 3D printing. And the tensile modulus of the scaffolds with oriented collagen fibers was nine times higher than that of the scaffolds with non-oriented fibers. Moreover, the effects of oriented collagen fibers on the proliferation, differentiation and mineralization of MC3T3-E1 cells were studied by CCK-8 assay, live/dead cell staining, alkaline phosphatase activity test, and Alizarin red staining. The results indicated that cell proliferation, differentiation and mineralization were significantly promoted by oriented collagen fibers, and the cells proliferated directionally in the direction of the fibers. Taken together, mineralized collagen fiber scaffolds with oriented collagen fibers have great potential in bone tissue engineering applications.

A Novel Collagen Aerogel with Relevant Features for Topical Biomedical Applications.

Collagen-based aerogels have great potential for topical biomedical applications. Collagen's natural affinity with skin, biodegradability, and gelling behavior are compelling properties to combine with the structural integrity of highly porous matrices in the dry form (aerogels). This work aimed to produce a novel collagen-based aerogel and to perform the material's solid-state and physicochemical characterization. Aerogels were obtained by performing different solvent exchange approaches of a collagen-gelled extract and drying the obtained alcogels with supercritical CO2. The resulting aerogels showed a sponge-like structure with a relatively dense mesoporous network with a specific surface area of 201-203 m2/g, a specific pore volume of 1.08-1.15 cm3/g, and a mean pore radius of ca. 14.7 nm. Physicochemical characterization confirmed that the obtained aerogels are composed of pure collagen, and the aerogel production process does not impact protein tertiary structure. Finally, the material swelling behavior was assessed at various pH values (4, 7, and 10). Collagen aerogels presented a high water uptake capacity up to ~2700 wt. %, pH-dependent stability, and swelling behavior in aqueous media. The results suggest that this collagen aerogel could be a promising scaffold candidate for topical biomedical applications.

Fibrillogenesis in collagen hydrogels accelerated by carboxylated microbeads

Collagen type I is a material widely used for 3D cell culture and tissue engineering. Different architectures, such as gels, sponges, membranes, and nanofibers, can be fabricated with it. In collagen hydrogels, the formation of fibrils and fibers depends on various parameters, such as the source of collagen, pH, temperature, concentration, age, etc. In this work, we study the fibrillogenesis process in collagen type I hydrogels with different types of microbeads embedded, using optical techniques such as turbidity assay and confocal reflectance microscopy. We observe that microbeads embedded in the collagen matrix hydrogels modify the fibrillogenesis. Our results show that carboxylated fluorescent microbeads accelerate 3.6 times the gelation, while silica microbeads slow down the formation of collagen fibrils by a factor of 1.9, both compared to pure collagen hydrogels. Our observations suggest that carboxylate microbeads act as nucleation sites and the early collagen fibrils bind to the microbeads.

Development of a Static Avascular and Dynamic Vascular Human Skin Equivalent Employing Collagen/Keratin Hydrogels.

One of the primary complications in generating physiologically representative skin tissue is the inability to integrate vasculature into the system, which has been shown to promote the proliferation of basal keratinocytes and consequent keratinocyte differentiation, and is necessary for mimicking representative barrier function in the skin and physiological transport properties. We created a 3D vascularized human skin equivalent (VHSE) with a dermal and epidermal layer, and compared keratinocyte differentiation (immunomarker staining), epidermal thickness (H&E staining), and barrier function (transepithelial electrical resistance (TEER) and dextran permeability) to a static, organotypic avascular HSE (AHSE). The VHSE had a significantly thicker epidermal layer and increased resistance, both an indication of increased barrier function, compared to the AHSE. The inclusion of keratin in our collagen hydrogel extracellular matrix (ECM) increased keratinocyte differentiation and barrier function, indicated by greater resistance and decreased permeability. Surprisingly, however, endothelial cells grown in a collagen/keratin extracellular environment showed increased cell growth and decreased vascular permeability, indicating a more confluent and tighter vessel compared to those grown in a pure collagen environment. The development of a novel VHSE, which incorporated physiological vasculature and a unique collagen/keratin ECM, improved barrier function, vessel development, and skin structure compared to a static AHSE model.

Exploring the potential of selective oxidation in bioconjugation of collagen with xyloglucan carboxylates

Xyloglucan (XG), as a natural biopolymer, possesses a sound biocompatibility and an impressive biodegradability, which are usually featured with abundant hydroxyl groups available for the bioconjugation with a bioactive moiety, suggesting a promising or unique value possibly applied in the field of biomedicine. In this study, XG was extracted from Tamarind seeds and subjected to four regioselective oxidation methods to introduce carboxyl groups onto the XG molecules for a bioconjugation with collagen. Galactose oxidase and reducing end aldehyde group oxidation mainly resulted in a low carboxylate content at ∼0.34 mmol/g, whereas the primary and secondary hydroxyl group oxidations would lead to a high carboxyl content at ∼0.84 mmol/g. The number-average molar mass (Mn) and weight-average molar mass (Mw) of XG were 8.8 × 105 g/mol and 1.1 × 106 g/mol, respectively. The oxidized XGs were then subjected to a further biofunctionalization with the collagen through EDC/NHS coupling, which exhibited a degree of conjugation rate, ranged from 50 % to 72 %. The collagen-conjugated at the C6 position of XGs exhibited the highest cell viability recorded at 168 % in promoting cell growth and proliferation after 72 h of culture, surpassing that of pure collagen recorded at 138 %, which may indeed suggest a promising value in a biomedical application.

Dialdehyde Starch as a Cross-Linking Agent Modifying Fish Collagen Film Properties.

The aim of this research was the modification of fish collagen films with various amounts of dialdehyde starch (DAS). Film properties were examined before and after the cross-linking process by DAS. Prepared biopolymer materials were characterized by Fourier Transform Infrared Spectroscopy and Atomic Force Microscopy. Moreover, the mechanical, thermal and swelling properties of the films were evaluated and the contact angle was measured. Research has shown that dialdehyde starch applied as a cross-linking agent influences collagen film properties. Mechanical testing indicated a decrease in Young's Modulus and an increase in breaking force, elongation at break, and tensile strength parameters. Results for contact angle were significantly higher for collagen films cross-linked with DAS; thus, the hydrophilicity of samples decreased. Modified samples presented a lower swelling degree in PBS than native collagen films. However, the highest values for the degree of swelling among the modified specimens were obtained from the 1% DAS samples, which were 717% and 702% for 1% and 2% collagen, respectively. Based on AFM images and roughness values, it was noticed that DAS influenced collagen film surface morphology. The lowest value of Rq was observed for 2%Coll_2%DAS and was approximately 10 nm. Analyzing thermograms for collagen samples, it was observed that pure collagen samples were less thermally stable than cross-linked ones. Dialdehyde starch is a promising cross-linking agent for collagen extracted from fish skin and may increase its applicability.

Bionanofactory for green synthesis of collagen nanoparticles, characterization, optimization, in-vitro and in-vivo anticancer activities

Collagen nanoparticles (collagen-NPs) are promising biological polymer nanoparticles due to their exceptional biodegradability and biocompatibility. Collagen-NPs were bio-fabricated from pure marine collagen using the cell-free supernatant of a newly isolated strain, Streptomyces sp. strain NEAA-3. Streptomyces sp. strain NEAA-3 was identified as Streptomyces plicatus strain NEAA-3 based on its cultural, morphological, physiological properties and 16S rRNA sequence analysis. The sequence data has been deposited under accession number OR501412.1 in the GenBank database. The face-centered central composite design (FCCD) was used to improve collagen-NPs biosynthesis. The maximum yield of collagen-NPs was 9.33 mg/mL with a collagen concentration of 10 mg/mL, an initial pH of 7, an incubation time of 72 h, and a temperature of 35 °C. Using the desirability function approach, the collagen-NPs biosynthesis obtained after FCCD optimization (9.53 mg/mL) was 3.92 times more than the collagen-NPs biosynthesis obtained before optimization process (2.43 mg/mL). The TEM analysis of collagen-NPs revealed hollow sphere nanoscale particles with an average diameter of 33.15 ± 10.02 nm. FTIR spectra confirmed the functional groups of the collagen, collagen-NPs and the cell-free supernatant that are essential for the efficient capping of collagen-NPs. The biosynthesized collagen-NPs exhibited antioxidant activity and anticancer activity against HeP-G2, MCF-7 and HCT116 cell lines. Collagen-NPs assessed as an effective drug loading carrier with methotrexate (MTX), a chemotherapeutic agent. The TEM analysis revealed that the average size of MTX-loaded collagen-NPs was 35.4 ± 8.9 nm. The percentages of drug loading (DL%) and encapsulation efficiency (EE%) were respectively 22.67 and 45.81%.

Hydrophilic, Porous, Fiber-Reinforced Collagen-Based Membrane for Corneal Repair.

Collagen membrane with outstanding biocompatibility exhibits immense potential in the field of corneal repair and reconstruction, but the poor mechanical properties limit its clinical application. Polycaprolactone (PCL) is a biodegradable polymer widely explored for application in corneal reconstruction due to its excellent mechanical properties, biocompatibility, easy processability, and flexibility. In this study, a PCL/collagen composite membrane with reinforced mechanical properties is developed. The membrane has a strong composite structure with collagen by utilizing a porous and hydrophilic PCL scaffold, maintaining its integrity even after immersion. The suture retention and mechanical tests demonstrate that compared with the pure collagen membrane, the prepared membrane has a greater tensile strength and twice the modulus of elasticity. Further, the suture retention strength is improved by almost two times. In addition, the membrane remains fully intact on the implant bed in an in vitro corneal defect model. Moreover, the membrane can be tightly sutured to a rabbit corneal defect, progressively achieve epithelialization, and remain unchanged during observation. Overall, the PCL/collagen composite membrane is a promising candidate as a suturable corneal restoration material in clinical keratoplasty.

Co-assembled biomimetic fibrils from collagen and chitosan for performance-enhancing hemostatic dressing.

The development of safe and efficient hemostatic materials is medically important to prevent death due to trauma bleeding. Exploiting the synergistic effect between the D-periodic functional domain of collagen fibrils on platelet activation and cationic chitosan on erythrocyte aggregation is expected to develop performance-enhanced hemostatic materials. In this study, we prepared collagen fibrils and chitosan composite hemostatic materials by modulating the self-assembled bionic fibrillation of collagen with different degrees of deacetylation (DD, 50%, 70% and 85%) of chitosan. The findings indicated that chitosan promoted collagen self-assembly, with all the collagen fibrils demonstrating a typical D-periodical structure similar to that of the native collagen. Furthermore, the composite demonstrated enhanced structural integrity and procoagulant capacity along with good biocompatibility. Notably, the fibrillar composites with 70% DD of chitosan exhibited optimal mechanical properties, procoagulant activity, and adhesion of erythrocytes and platelets. Compared to pure collagen fibrils and the commercial hemostatic agent Celox™, the collagen/chitosan fibrillar composite treatment significantly accelerated hemostasis in the rat tail amputation model and liver injury model. This research offers new insights into the development of hemostatic materials and indicates that collagen-chitosan composites hold promising potential for clinical applications.

Collagen-Based Medical Devices for Regenerative Medicine and Tissue Engineering.

Assisted reproductive technologies are key to solving the problems of aging and organ defects. Collagen is compatible with living tissues and has many different chemical properties; it has great potential for use in reproductive medicine and the engineering of reproductive tissues. It is a natural substance that has been used a lot in science and medicine. Collagen is a substance that can be obtained from many different animals. It can be made naturally or created using scientific methods. Using pure collagen has some drawbacks regarding its physical and chemical characteristics. Because of this, when collagen is processed in various ways, it can better meet the specific needs as a material for repairing tissues. In simpler terms, collagen can be used to help regenerate bones, cartilage, and skin. It can also be used in cardiovascular repair and other areas. There are different ways to process collagen, such as cross-linking it, making it more structured, adding minerals to it, or using it as a carrier for other substances. All of these methods help advance the field of tissue engineering. This review summarizes and discusses the current progress of collagen-based materials for reproductive medicine.

Development of a highly concentrated collagen ink for the creation of a 3D printed meniscus

The most prevalent extracellular matrix (ECM) protein in the meniscus is collagen, which controls cell activity and aids in preserving the biological and structural integrity of the ECM. To create stable and high-precision 3D printed collagen scaffolds, ink formulations must possess good printability and cytocompatibility. This study aims to overlap the limitation in the 3D printing of pure collagen, and to develop a highly concentrated collagen ink for meniscus fabrication. The extrusion test revealed that 12.5 % collagen ink had the best combination of high collagen concentration and printability. The ink was specifically designed to have load-bearing capacity upon printing and characterized with respect to rheological and extrusion properties. Following printing of structures with different infill, a series of post-processing steps, including salt stabilization, pH shifting, washing, freeze-drying, crosslinking and sterilization were performed, and optimised to maintain the stability of the engineered construct. Mechanical testing highlighted a storage modulus of 70 kPa for the lower porous structure while swelling properties showed swelling ratio between 9 and 11 after 15 min of soaking. Moreover, human avascular and vascular meniscus cells cultured on the scaffolds deposited a meniscus-like matrix containing collagen I, II and glycosaminoglycans after 28 days of culture. Finally, as proof-of-concept, human size 3D printed meniscus scaffold were created.

Guided bone regeneration in long-bone defect with a bilayer mineralized collagen membrane

Bone regeneration for large, critical-sized bone defects remains a clinical challenge nowadays. Guided bone regeneration (GBR) is a promising technique for the repair of multiple bone defects, which is widely used in oral and maxillofacial bone defects but is still unsatisfied in the treatment of long bone defects. Here, we successfully fabricated a bilayer mineralized collagen/collagen (MC/Col)-GBR membrane with excellent osteoinductive and barrier function by coating the MC particles prepared via in situ biomimetic mineralization process on one side of a sheet-like pure collagen layer. The aim of the present study was to investigate the physicochemical properties and biological functions of the MC/Col film, and to further evaluate its bone regeneration efficiency in large bone defect repair. Fourier-transform infrared spectra and X-ray diffraction patterns confirmed the presence of both hydroxyapatite and collagen phase in the MC/Col film, as well as the chemical interaction between them. stereo microscope, scanning electron microscopy and atomic force microscope showed the uniform distribution of MC particles in the MC/Col film, resulting in a rougher surface compared to the pure Col film. The quantitative analysis of surface contact angle, light transmittance and tensile strength demonstrated that the MC/Col film have better hydrophilicity, mechanical properties, light-barrier properties, respectively. In vitro macrophage co-culture experiments showed that the MC/Col film can effectively inhibit macrophage proliferation and fusion, reducing fibrous capsule formation. In vivo bone repair assessment of a rabbit critical segmental radial defect proved that the MC/Col film performed better than other groups in promoting bone repair and regeneration due to their unique dual osteoinductive/barrier function. These findings provided evidence that MC/Col film has a great clinical potential for effective bone defect repair.Graphic abstract