Implants of polyanionic collagen matrix in bone defects of ovariectomized rats

In recent years, there has been a great interest in the development of biomaterials that could be used in the repair of bone defects. Collagen matrix (CM) has the advantage that it can be modified chemically to improve its mechanical properties. The aim of the present study was to evaluate the effect of three-dimensional membranes of native or anionic (submitted to alkaline treatment for 48 or 96 h) collagen matrix on the consolidation of osteoporosis bone fractures resulting from the gonadal hormone alterations caused by ovariectomy in rats subjected to hormone replacement therapy. The animals received the implants 4 months after ovariectomy and were sacrificed 8 weeks after implantation of the membranes into 4-mm wide bone defects created in the distal third of the femur with a surgical bur. Macroscopic analysis revealed the absence of pathological alterations in the implanted areas, suggesting that the material was biocompatible. Microscopic analysis showed a lower amount of bone ingrowth in the areas receiving the native membrane compared to the bone defects filled with the anionic membranes. In ovariectomized animals receiving anionic membranes, a delay in bone regeneration was observed mainly in animals not subjected to hormone replacement therapy. We conclude that anionic membranes treated with alkaline solution for 48 and 96 h presented better results in terms of bone ingrowth.     

Chitosan-based polyelectrolyte complex scaffolds with antibacterial properties for treating dental bone defects

The aim of this study was to develop an antibacterial polyelectrolyte complex (PEC) scaffold for treating dental bone defects. The PEC scaffold was composed of chitosan (CS), γ-polyglutamic acid (γ-PGA), and carboxy-methyl-cellulose (CMC). The resulted network structures formed via electrostatic crosslinking were characterized by using FTIR, gel content, equilibrium swelling ratio, volume change, and SEM test. The antibacterial property, cell cytotoxicity, and in vivo biocompatibility tests were conducted according to an agar diffusion method, ISO10993-5, and ISO 10993-6, respectively. The resulted specimens showed an interconnected pore structure with pore sizes ranging 100-500 μm. The equilibrium swelling ratio, volume change, and antibacterial property were inversely proportional to the gel content. The PEC-2 scaffold composed of 8 wt.% CS, and 2 wt.% γ-PGA + 2 wt.% CMC had more-suitable gel properties (gel content of 55.3 ± 1.1 wt.% and volume change of 97.7 ± 1.4 v/v%) with inhibition zones of 14.4 ± 0.3 mm for Escherichia coli and 13.0 ± 0.7 mm for Staphylococcus aureus. The cytotoxicity and cell attachment tests of the PEC scaffolds showed satisfactory cell compatibility. Moreover, the in vivo biocompatibility test of the PEC scaffolds revealed little foreign body reaction. For this reason, the newly developed antibacterial PEC scaffold may be a good alternative for dental applications.     

Nanostructured 3-D collagen/nanotube biocomposites for future bone regeneration scaffolds

The field of bionanotechnology has been rapidly growing during the last few years and we can now envision a controllable integration between biological and artificial matter, where new biomimetic structures with a wide range of chemical and physical properties will promote the development of a novel generation of medical devices. In this work we describe a collagen/carbon nanotube composite which has the potential to be used as a scaffold for tissue regeneration. Because this biocomposite incorporates the advantageous properties of both collagen and carbon nanotubes, it has most of the characteristics that an ideal biomaterial requires in order to be used as an osteoinductive agent. This biocomposite is bioresorbable and biodegradable and has the desired mechanical rigidity while maintaining a three-dimensional(3-D) nanostructured surface. Tuned stability and swelling were achieved under fluid environments by varying the amount of carbon nanotubes (CNTs) incorporated into the composite. These variations can dictate the degree of interaction between fibroblastic cells and the biomaterials. Proof-of-concept was shown by performing an in vitro induced mineralization of hydroxylapatite crystals under physiological conditions. Furthermore, the ability to attach biofunctional groups to the CNT walls can open a new road for tissue regeneration since the combination of CNTs with specific growth factors or cellular ligands can create an environment capable of signaling and influencing specific cell functions. Our observations suggest that collagen/carbon nanotube biocomposites will have important uses in a wide range of biotechnological areas. Electronic Supplementary Material  Supplementary material is available for this article at and is accessible for authorized users.     

Restoration of mandibular bone defects with demineralized bone matrix combined with three-dimensional cultured bone marrow-derived mesenchymal stem cells in minipig models

Mandibular defects, caused by congenital, pathological or iatrogenic insults, can significantly affect patient quality of life. The reconstruction of mandible has recently gained the interest of clinical and tissue engineering researchers. The purpose of this study was to evaluate the effectiveness of three-dimensional (3-D) cultured autologous grafts prepared using bone marrow-derived mesenchymal stem cells (BMSCs) combined with demineralized bone matrix (DBM) scaffolds for the restoration of mandibular defects. Cylindrical defects were created in the mandibular body of minipigs and filled with 3D-cultured BMSCs/DBM autografts, 2D-cultured BMSCs/DBM autografts, DBM material (without cells), or were left unfilled (blank). Using computed tomographic (CT) imaging and histological staining, we found that treatment of mandibular defects using 3-D cultured BMSCs/DBM autografts offered improvements in bone formation over both 2-D cultured autografts and cell-free DBM scaffolds. We found increased osteoid formation in 3D and 2D cultures, with more osteogenic cells present in the 3D constructs. We suggest that 3-D cultured homograft BMSCs combined with DBM scaffolds represents a new strategy for bone reconstruction, with potential future clinical applicability.     

Effect of multiple unconfined compression on cellular dense collagen scaffolds for bone tissue engineering

Plastic compression of hydrated collagen gels rapidly produces biomimetic scaffolds of improved mechanical properties. These scaffolds can potentially be utilised as cell seeded systems for bone tissue engineering. This work investigated the influence of multiple unconfined compression on the biocompatibility and mechanical properties of such systems. Single and double compressed dense collagen matrices were produced and characterised for protein dry weight, morphology and mechanical strength. Compression related maintenance of the seeded HOS TE85 cell line viability in relation to the extent of compression was evaluated up to 10 days in culture using the TUNEL assay. Fluorescence Live/Dead assay was conducted to examine overall cell survival and morphology. Cell induced structural changes in the dense collagenous scaffolds were assessed by routine histology. The mechanical properties of the cellular scaffolds were also evaluated as a function of time in culture. It is clear that a single plastic compression step produced dense collagenous scaffolds capable of maintaining considerable cell viability and function as signs of matrix remodeling, and maintenance of mechanical properties were evident. Such scaffolds should therefore be further developed as systems for bone tissue regeneration.     

Comparative study on the role of gelatin,chitosan and their combination as tissue engineered scaffolds on healing and regeneration of critical sized bone defects: an in vivo study

Gelatin and chitosan are natural polymers that have extensively been used in tissue engineering applications. The present study aimed to evaluate the effectiveness of chitosan and gelatin or combination of the two biopolymers (chitosan–gelatin) as bone scaffold on bone regeneration process in an experimentally induced critical sized radial bone defect model in rats. Fifty radial bone defects were bilaterally created in 25 Wistar rats. The defects were randomly filled with chitosan, gelatin and chitosan–gelatin and autograft or left empty without any treatment (n?=?10 in each group). The animals were examined by radiology and clinical evaluation before euthanasia. After 8?weeks, the rats were euthanized and their harvested healing bone samples were evaluated by radiology, CT-scan, biomechanical testing, gross pathology, histopathology, histomorphometry and scanning electron microscopy. Gelatin was biocompatible and biodegradable in vivo and showed superior biodegradation and biocompatibility when compared with chitosan and chitosan–gelatin scaffolds. Implantation of both the gelatin and chitosan–gelatin scaffolds in bone defects significantly increased new bone formation and mechanical properties compared with the untreated defects (P?<?0.05). Combination of the gelatin and chitosan considerably increased structural and functional properties of the healing bones when compared to chitosan scaffold (P?<?0.05). However, no significant differences were observed between the gelatin and gelatin–chitosan groups in these regards (P?>?0.05). In conclusion, application of the gelatin alone or its combination with chitosan had beneficial effects on bone regeneration and could be considered as good options for bone tissue engineering strategies. However, chitosan alone was not able to promote considerable new bone formation in the experimentally induced critical-size radial bone defects.     

Elastin and collagen enhances electrospun aligned polyurethane as scaffolds for vascular graft

Mismatch in mechanical properties between synthetic vascular graft and arteries contribute to graft failure. The viscoelastic properties of arteries are conferred by elastin and collagen. In this study, the mechanical properties and cellular interactions of aligned nanofibrous polyurethane (PU) scaffolds blended with elastin, collagen or a mixture of both proteins were examined. Elastin softened PU to a peak stress and strain of 7.86 MPa and 112.28 % respectively, which are similar to those observed in blood vessels. Collagen-blended PU increased in peak stress to 28.14 MPa. The growth of smooth muscle cells (SMCs) on both collagen-blended and elastin/collagen-blended scaffold increased by 283 and 224 % respectively when compared to PU. Smooth muscle myosin staining indicated that the cells are contractile SMCs which are favored in vascular tissue engineering. Elastin and collagen are beneficial for creating compliant synthetic vascular grafts as elastin provided the necessary viscoelastic properties while collagen enhanced the cellular interactions.     

Repair of rabbit radial bone defects using true bone ceramics combined with BMP-2-related peptide and type I collagen

An ideal bone graft material is the one characterized with good biocompatibility, biodegradation, osteoconductivity and osteoinductivity. In this study, a novel synthetic BMP-2-related peptide (designated P24) corresponding to residues of the knuckle epitope of BMP-2 was introduced into a biomimetic scaffold based on sintered bovine bone or true bone ceramics (TBC) and type I collagen (TBC/collagen I) using a simulated body fluid (SBF). Hydroxylapatite crystal mineralization with a Ca/P molar ratio of 1.63 was observed on the surface of P24/TBC/collagen I composite by scanning electron microscopy (SEM), energy dispersive X-ray spectroscopy (EDX) and X-ray diffraction (XRD) techniques. Cell adhesion rate evaluation of bone marrow stromal cells (BMSCs) seeded on materials in vitro showed that the percentage of cells attached to P24/TBC/collagen I composite was significantly higher than that of the TBC/collagen I composite. A 10 mm unilateral segmental bone defect was created in the radius of New Zealand white rabbits and randomly implanted with three groups of biomaterials (Group A: P24/TBC/collagen I composite; Group B: TBC/collagen I composite and Group C: TBC alone). Based on radiographic evaluation and histological examination, the implants of P24/TBC/collagen I composite significantly stimulated bone growth, thereby confirming the enhanced rate of bone healing compared with that of TBC/collagen I composite and TBC alone. It was concluded that BMP-2-related peptide P24 could induce nucleation of calcium phosphate crystals on the surface of TBC/collagen I composite. The TBC/collagen I composite loaded with the synthetic BMP-2-related peptide is a promising scaffold biomaterial for bone tissue engineering.     

Alginate hydrogels crosslinked with different strontium-calcium ratios as injectable scaffolds for bone tissue engineering

The easy loss of crosslinking ions in alginate can result in a structural collapse of the physiological environment, thereby losing its characteristics as a bone scaffold. Meanwhile, alginate lacks osteoconductive properties, which are necessary for ideal bone scaffolds. In this study, strontium (Sr) in combination with calcium (Ca) at different ratios were used as a crosslinking agent for the alginate to investigate the effect of Ca–Sr ratio on the physicochemical properties and biological preformation of alginate hydrogel. Here, Ca and Sr in different weight ratios (4:0, 3:1, 2:2, 1:3, and 0:4) were employed as crosslinking agents. The physicochemical properties of hydrogels, including pore size, elastic modulus, degradation rate and swelling ratio, could be effectively tuned by controlling the amount of Sr. The ion release experiment revealed a burst release of Sr²⁺ in the first day after crosslinking. However, after 3 days, the amount of Sr²⁺ release had significantly declined and was proportional to the total strontium initially introduced into the alginate. Meanwhile, the live/dead results exhibited higher cell viability for alginate with 2:2 Ca–Sr weight ratio. The alginate with 2:2 Ca–Sr ratio not only improved osteoblastic attachment, but also up-regulated the alkaline phosphatase activity, the expression of osteogenic marker genes, and relative growth factors. These findings indicate that alginate with 2:2 Ca–Sr ratio might be a promising scaffold for bone tissue engineering.

Graphical abstract

     

Controlling stem cell behavior with decellularized extracellular matrix scaffolds

Decellularized tissues have become a common regenerative medicine platform with multiple materials being researched in academic laboratories, tested in animal studies, and used clinically. Ideally, when a tissue is decellularized the native cell niche is maintained with many of the structural and biochemical cues that naturally interact with the cells of that particular tissue. This makes decellularized tissue materials an excellent platform for providing cells with the signals needed to initiate and maintain differentiation into tissue-specific lineages. The extracellular matrix (ECM) that remains after the decellularization process contains the components of a tissue specific microenvironment that is not possible to create synthetically. The ECM of each tissue has a different composition and structure and therefore has unique properties and potential for affecting cell behavior. This review describes the common methods for preparing decellularized tissue materials and the effects that decellularized materials from different tissues have on cell phenotype.     

Iron nanoparticles from blood coated with collagen as a matrix for synthesis of nanohydroxyapatite

A simple wet precipitation technique was used to prepare nanobiocomposite containing iron nanoparticles coated with collagen. This nanobiocomposite was used as matrix for the synthesis of nanohydroxyapatite. The physicochemical characteristic studies of the nanohydroxyapatite thus formed were carried out using fourier transform infrared spectroscopy, transmission electron microscopy, scanning electron microscopy, energy-dispersive X-ray spectroscopy and X-ray diffraction technique to confirm the formation of hydroxyapatite on iron nanoparticle–collagen complex. The results of the above studies supported the formation of iron nanoparticle–collagen–hydroxyapatite composite. The biological studies such as biocompatibility and hemocompatibility were carried out for nanohydroxyapatite using different cell lines and blood sample. The results of biocompatibility and hemolytic assay revealed that the prepared nanobiocomposite was 100 % biocompatible and hemocompatible. This nanobiocomposite may be used for biomedical application such as injectables for targeted delivery and as scaffold for tissue engineering.     

Innovative collagen nano-hydroxyapatite scaffolds offer a highly efficient non-viral gene delivery platform for stem cell-mediated bone formation

The ability of nano-hydroxyapatite (nHA) particles developed in-house to act as non-viral delivery vectors is assessed. These nHA particles are combined with collagen to yield bioactive, biodegradable collagen nano-hydroxyapatite (coll-nHA) scaffolds. Their ability to act as gene-activated matrices for BMP2 delivery is demonstrated with successful transfection of mesenchymal stem cells (MSCs) resulting in high calcium production.     

Effect of expanded bone marrow-derived osteoprogenitor cells seeded into polycaprolactone/tricalcium phosphate scaffolds in new bone regeneration of rabbit mandibular defects

The purpose of this study was to assess and evaluate new bone formation in rabbit marginal mandibular defects using expanded bone marrow-derived osteoprogenitor cells seeded in three-dimensional scaffolds of polycaprolactone/tricalcium phosphate (PCL/TCP). Bone marrow was harvested from the rabbit ilium and rabbit bone marrow-derived osteoprogenitor cells were isolated and expanded in standard culture medium and osteogenic medium supplement. The cells were then seeded into the PCL/TCP scaffolds and the cell/scaffold constructions were implanted into prepared defects in rabbit mandibles. PCL/TCP scaffold alone and autogenous bone graft from the mandible were also implanted into the other prepared defects. The specimens were evaluated at 4 and 8 weeks after the implantation using clinical, radiographic, and histological techniques. The results of the experimental group demonstrated more newly formed bone on the surface and in the pores of the PCL/TCP scaffolds. In addition, the osteoblasts, osteocytes, and new bone trabeculae were identified throughout the defects that were implanted with the cell/scaffold constructions. The PCL/TCP alone group was filled mostly with fibrous cells particularly in the middle region with less bone formation. These results would suggest that the derived osteotoprogenitor cells have the potential to form bone tissue when seeded onto PCL/TCP scaffolds.     

Hydroxyapatite/gelatin/gellan sponges as nanocomposite scaffolds for bone reconstruction

The aim of this work was the morphological, physicochemical, mechanical and biological characterization of a new composite system, based on gelatin, gellan and hydroxyapatite, and mimicking the composition of natural bone. Porous scaffolds were prepared by freeze–drying technique, under three different conditions of freezing. The morphological analysis showed a homogeneous porosity, with well interconnected pores, for the sample which underwent a more rapid freezing. The elastic modulus of the same sample was close to that of the natural bone. The presence of interactions among the components was demonstrated through the physicochemical investigation. In addition, the infrared chemical imaging analysis pointed out the similarity among the composite scaffold and the natural bone, in terms of chemical composition, homogeneity, molecular interactions and structural conformation. Preliminary biological characterization showed a good adhesion and proliferation of human mesenchymal stem cells.     

Coating of polyurethane scaffolds with collagen: comparison of coating and cross-linking techniques

Collagen has been coated successfully onto numerous hydrophilic polymer scaffolds to improve cell adhesion. Due to the hydrophobic nature of thermoplastic polyurethane (TPU), coating with aqueous collagen solution is problematic for such scaffolds. This study facilitated the coating of TPU with collagen and compared cross-linking and coating techniques. Three different cross-linking methods were compared. Both thermal and glutaraldehyde methods showed proof of cross-linking; however glutaraldehyde seemed to be superior to the other methods. The use of human urine as a wetting agent and the chemical glutaraldehyde had no effect on a cytotoxicity test performed by means of a WST-1 assay with a fibroblastic cell line. Three different coating techniques for porous TPU scaffolds were also investigated: ultrasound, pressurized air and injection. Of these, injection performed best. This method facilitated a coating of 100% of the porous scaffolds examined, which was verified by staining, FTIR and SEM.