Fabrication of 3D melt electrowritting multiphasic scaffold with bioactive and osteoconductivite functionalities for periodontal regeneration

This work aimed to design a multifunctional biphasic 3D scaffold for periodontal tissue regeneration. A 3D fibrous scaffold made from medical-grade poly (ε-caprolactone) (PCL) with high porosity (>90%) and well-oriented fibres was fabricated by a custom design melt electrowriting (MEW) device. A biomimetic process was employed to form a bioactive calcium phosphate (CaP) layer with nanostructure (nanoflakes-like) morphology onto the 3D MEW fibrous surface to stimulate rapid bone formation. Primary human osteoblasts (hOBs) were seeded within the coated 3D fibrous scaffolds for 28 days to acquire the bone compartment of the tissue-engineered construct (TEC). The biphasic construct was obtained by placing an established in vitro periodontal ligament (PDL) cell sheet onto the surface of the bone compartment. Subsequently, a decellularized multiphasic TEC by exploiting a lyophilization approach was obtained. Laser scanning confocal microscopy and scanning electron microscopy confirmed the retention of a functional extracellular matrix within the PDL and bone compartments following scaffold decellularization and lyophilization processes. These findings suggest that lyophilized decellularized biphasic 3D constructs with high porosity constitute a viable ‘off the shelf’ strategy for developing an extracellular matrix-based product to facilitate periodontal regeneration.     

Metformin-Incorporated Gelatin/Nano-Hydroxyapatite Scaffolds Promotes Bone Regeneration in Critical Size Rat Alveolar Bone Defect Model

In this study, we fabricated gelatin/nano-hydroxyapatite/metformin scaffold (GHMS) and compared its effectiveness in bone regeneration with extraction-only, Sinbone, and Bio-Oss Collagen^® groups in a critical size rat alveolar bone defect model. GHMS was synthesized by co-precipitating calcium hydroxide and orthophosphoric acid within gelatin solution, incorporating metformin, and cross-linked by microbial transglutaminase. The morphology, characterization, and biocompatibility of scaffold were examined. The in vitro effects of GHMS on osteogenic gene and protein expressions were evaluated. In vivo bone formation was assessed in a critical size rat alveolar bone defect model with micro-computed tomography and histological examination by comparing GHMS with extraction-only, Sinbone, and Bio-Oss Collagen^®. The synthesized GHMS had a highly interconnected porous structure with a mean pore size of 81.85 ± 13.8 µm. GHMS exhibited good biocompatibility; promoted ALPL, RUNX2, SP7, BGLAP, SPARC and Col1a1 gene expressions; and upregulated the synthesis of osteogenic proteins, including osteonectin, osteocalcin, and collagen type I. In critical size rat alveolar bone defects, GHMS showed superior bone regeneration compared to extraction-only, Sinbone, and Bio-Oss Collagen^® groups as manifested by greater alveolar ridge preservation, while more bone formation with a lower percentage of connective tissue and residual scaffold at the defect sites grafted with GHMS in histological staining. The GHMS presented in this study may be used as a potential bone substitute to regenerate alveolar bone. The good biocompatibility, relatively fast degradation, interconnected pores allowing vascularization, and higher bioactivity properties of the components of the GHMS (gelatin, nHA, and metformin) may contribute to direct osteogenesis.     

Superior Alignment of Human iPSC-Osteoblasts Associated with Focal Adhesion Formation Stimulated by Oriented Collagen Scaffold

Human-induced pluripotent stem cells (hiPSCs) can be applied in patient-specific cell therapy to regenerate lost tissue or organ function. Anisotropic control of the structural organization in the newly generated bone matrix is pivotal for functional reconstruction during bone tissue regeneration. Recently, we revealed that hiPSC-derived osteoblasts (hiPSC-Obs) exhibit preferential alignment and organize in highly ordered bone matrices along a bone-mimetic collagen scaffold, indicating their critical role in regulating the unidirectional cellular arrangement, as well as the structural organization of regenerated bone tissue. However, it remains unclear how hiPSCs exhibit the cell properties required for oriented tissue construction. The present study aimed to characterize the properties of hiPSCs-Obs and those of their focal adhesions (FAs), which mediate the structural relationship between cells and the matrix. Our in vitro anisotropic cell culture system revealed the superior adhesion behavior of hiPSC-Obs, which exhibited accelerated cell proliferation and better cell alignment along the collagen axis compared to normal human osteoblasts. Notably, the oriented collagen scaffold stimulated FA formation along the scaffold collagen orientation. This is the first report of the superior cell adhesion behavior of hiPSC-Obs associated with the promotion of FA assembly along an anisotropic scaffold. These findings suggest a promising role for hiPSCs in enabling anisotropic bone microstructural regeneration.     

Enhanced Regeneration of Vascularized Adipose Tissue with Dual 3D-Printed Elastic Polymer/dECM Hydrogel Complex

A flexible and bioactive scaffold for adipose tissue engineering was fabricated and evaluated by dual nozzle three-dimensional printing. A highly elastic poly (L-lactide-co-ε-caprolactone) (PLCL) copolymer, which acted as the main scaffolding, and human adipose tissue derived decellularized extracellular matrix (dECM) hydrogels were used as the printing inks to form the scaffolds. To prepare the three-dimensional (3D) scaffolds, the PLCL co-polymer was printed with a hot melting extruder system while retaining its physical character, similar to adipose tissue, which is beneficial for regeneration. Moreover, to promote adipogenic differentiation and angiogenesis, adipose tissue-derived dECM was used. To optimize the printability of the hydrogel inks, a mixture of collagen type I and dECM hydrogels was used. Furthermore, we examined the adipose tissue formation and angiogenesis of the PLCL/dECM complex scaffold. From in vivo experiments, it was observed that the matured adipose-like tissue structures were abundant, and the number of matured capillaries was remarkably higher in the hydrogel–PLCL group than in the PLCL-only group. Moreover, a higher expression of M2 macrophages, which are known to be involved in the remodeling and regeneration of tissues, was detected in the hydrogel–PLCL group by immunofluorescence analysis. Based on these results, we suggest that our PLCL/dECM fabricated by a dual 3D printing system will be useful for the treatment of large volume fat tissue regeneration.     

Hesperidin Promotes Osteogenesis and Modulates Collagen Matrix Organization and Mineralization In Vitro and In Vivo

This study evaluated the direct effect of a phytochemical, hesperidin, on pre-osteoblast cell function as well as osteogenesis and collagen matrix quality, as there is little known about hesperidin’s influence in mineralized tissue formation and regeneration. Hesperidin was added to a culture of MC3T3-E1 cells at various concentrations. Cell proliferation, viability, osteogenic gene expression and deposited collagen matrix analyses were performed. Treatment with hesperidin showed significant upregulation of osteogenic markers, particularly with lower doses. Mature and compact collagen fibrils in hesperidin-treated cultures were observed by picrosirius red staining (PSR), although a thinner matrix layer was present for the higher dose of hesperidin compared to osteogenic media alone. Fourier-transform infrared spectroscopy indicated a better mineral-to-matrix ratio and matrix distribution in cultures exposed to hesperidin and confirmed less collagen deposited with the 100-µM dose of hesperidin. In vivo, hesperidin combined with a suboptimal dose of bone morphogenetic protein 2 (BMP2) (dose unable to promote healing of a rat mandible critical-sized bone defect) in a collagenous scaffold promoted a well-controlled (not ectopic) pattern of bone formation as compared to a large dose of BMP2 (previously defined as optimal in healing the critical-sized defect, although of ectopic nature). PSR staining of newly formed bone demonstrated that hesperidin can promote maturation of bone organic matrix. Our findings show, for the first time, that hesperidin has a modulatory role in mineralized tissue formation via not only osteoblast cell differentiation but also matrix organization and matrix-to-mineral ratio and could be a potential adjunct in regenerative bone therapies.     

Enhanced function of chondrocytes in a chitosan-based hydrogel to regenerate cartilage tissues by accelerating degradability of the hydrogel via a hydrolysable crosslinker

Chitosan-based hydrogels as scaffolds for culturing chondrocytes were prepared using linkers with and without hydrolysable poly(dl -lactide) (PLA) segments. The evaluation of the cultured chondrocytes in them indicated that the accelerated degradation of the hydrogel via hydrolysis of the PLA slightly promoted production of the sulfated glycosaminoglycan and drastically improved that of collagen from the encapsulated chondrocytes, which are the chondrospecific extracellular matrix components. Furthermore, the accelerated degradability significantly upregulated the gene expression for Collagen II production and downregulated that for Collagen I production of the encapsulated chondrocytes. Because major component of the articular cartilage tissue is Collagen II-rich hyaline cartilage, these results suggest the degradation of the scaffolds is an important parameter in cartilage tissue regeneration and the accelerated degradability may have benefits on promotion of cartilage tissue regeneration especially from the viewpoint of hyaline cartilage-like collagen production. © 2019 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2019 , 137, 48893.     

Interaction of human smooth muscle cells on random and aligned nanofibrous scaffolds of PHBV and PHBV-gelatin

Tissue-engineered scaffolds with nanofibrous morphology have been shown to be effective in regeneration of tissues because nanofibers mimic the native architecture of the extracellular matrix. The unique alignment in the native tissue motivated the authors to fabricate aligned nanofibers of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) and PHBV-gelatin. The in vitro potential of the scaffolds was evaluated using human smooth muscle cells. MTS study confirmed that PHBV aligned nanofibrous scaffold promotes better cell proliferation as well as gene expression of key contractile and extracellular matrix markers than their PHBV-gelatin counterparts. Hence, the PHBV aligned nanofibers can be used as a biomimetic scaffold for the regeneration of esophagus. Electrospinning system for aligned nanofibers fabrication (A) and interaction of human smooth muscle cells on aligned nanofibers (B).     

Calcined Hydroxyapatite with Collagen I Foam Promotes Human MSC Osteogenic Differentiation

Collagen I-based foams were modified with calcined or noncalcined hydroxyapatite or calcium phosphates with various particle sizes and pores to monitor their effect on cell interactions. The resulting scaffolds thus differed in grain size, changing from nanoscale to microscopic, and possessed diverse morphological characteristics and resorbability. The materials’ biological action was shown on human bone marrow MSCs. Scaffold morphology was identified by SEM. Using viability test, qPCR, and immunohistochemical staining, we evaluated the biological activity of all of the materials. This study revealed that the most suitable scaffold composition for osteogenesis induction is collagen I foam with calcined hydroxyapatite with a pore size of 360 ± 130 µm and mean particle size of 0.130 µm. The expression of osteogenic markers RunX2 and ColI mRNA was promoted, and a strong synthesis of extracellular protein osteocalcin was observed. ColI/calcined HAP scaffold showed significant osteogenic potential, and can be easily manipulated and tailored to the defect size, which gives it great potential for bone tissue engineering applications.     

Bone Regeneration Guided by a Magnetized Scaffold in an Ovine Defect Model

The reconstruction of large segmental defects still represents a critical issue in the orthopedic field. The use of functionalized scaffolds able to create a magnetic environment is a fascinating option to guide the onset of regenerative processes. In the present study, a porous hydroxyapatite scaffold, incorporating superparamagnetic Fe₃O₄ nanoparticles (MNPs), was implanted in a critical bone defect realized in sheep metatarsus. Superparamagnetic nanoparticles functionalized with hyperbranched poly(epsilon-Lysine) peptides and physically complexed with vascular endothelial growth factor (VEGF) where injected in situ to penetrate the magnetic scaffold. The scaffold was fixed with cylindrical permanent NdFeB magnets implanted proximally, and the magnetic forces generated by the magnets enabled the capture of the injected nanoparticles forming a VEGF gradient in its porosity. After 16 weeks, histomorphometric measurements were performed to quantify bone growth and bone-to-implant contact, while the mechanical properties of regenerated bone via an atomic force microscopy (AFM) analysis were investigated. The results showed increased bone regeneration at the magnetized interface; this regeneration was higher in the VEGF-MNP-treated group, while the nanomechanical behavior of the tissue was similar to the pattern of the magnetic field distribution. This new approach provides insights into the ability of magnetic technologies to stimulate bone formation, improving bone/scaffold interaction.     

胶原和水解胶原蛋白的结构、性能和功效研究及应用前景

胶原蛋白是一类在所有哺乳动物中都存在的纤维状、大分子蛋白质,普遍分布于细胞外基质(ECM)中。它是动物体内细胞间隙中最重要,也是含量最多、最丰富的蛋白质,起着支撑器官保护机体的功能。胶原蛋白所持有的许多优良特性使其在各领域中的应用具有极大的潜质。水解胶原蛋白由于交联结构的解体和分子量大大降低使其溶解性和口服吸收利用率大大的提高。此外,胶原多肽还可以促进食品中的其它蛋白质的吸收。本综述对胶原及水解胶原蛋白的组成、结构、功效、应用和开发前景进行了研究和探讨。     

Poly(lactide-co-glycolide) nanoparticles embedded in a micropatterned collagen scaffold for neuronal tissue regeneration

Micropatterned collagen scaffold with axially oriented pores embedded with poly(lactide-co-glycolide) nanoparticles (PLGA NPs) was synthesized and characterized. Two different concentrations of PLGA nanoparticles have been tested and the experimental results indicate that the concentration affects the release kinetic, whereas the stiffness, the crosslink density, and the degradation rate of the collagen matrix are comparable to bare scaffold. Further, the proposed crosslinking procedure provides a resistance to thermal and enzymatic degradation, thereby promoting the persistence of scaffold for a period of time compatible with nerve regeneration.     

基于三维打印骨支架制备及其性能

利用三维打印（3DP）成型技术制备了羟基磷灰石（HAP）支架与混合壳聚糖（CS）的HAP-CS复合支架,并用Ⅰ型胶原蛋白（Collagen TypeⅠ）对HAP-CS支架进行了改性处理;通过抗压强度实验、孔隙率测定、微观形貌观察、亲水性及碱性磷酸酶（ALP）活性检测实验研究了所制备支架性能。结果表明添加CS可提高HAP支架抗压强度74.5%,但未降低支架孔隙率;经Collagen Type I改性的HAP-CS支架的亲水性有显著提高,ALP活性检测结果显示Collagen Type Ⅰ可提高细胞骨化能力。综上,本研究所制备的HAP-CS改性支架具有良好的性能及生物活性,可用于骨缺损修复研究。     

Fabrication and characterization of aligned nanofibrous PLGA/Collagen blends as bone tissue scaffolds

Aligned nanofibrous blends of poly (d, l-lactide-co-glycolide) (PLGA) and collagen with various PLGA/collagen compositions (80/20, 65/35 and 50/50) were fabricated by electrospinning and characterized for bone tissue engineering. Morphological characterization showed that the addition of collagen to PLGA resulted in narrowing of the diameter distribution and a reduction in average diameter. Differential scanning calorimetric (DSC) studies showed that the triple helix structure of the native collagen was not destroyed during the fabrication process. However, the blending had a marked effect on the overall enthalpy of the blends, whereby the total enthalpy decreased as the collagen content decreased. Thermogravimetric analysis showed the addition of collagen increased the hydrophilicity of the scaffolds. The crosslinking of collagen to increase the biostability was done using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) in ethanol and an overall ∼25% degree of crosslinking was achieved. The EDC crosslinking had little effect on the nanofibrous morphology of the 80/20 blend system; however, the nanofibrous features were compromised to some extent at higher collagen concentrations. The mechanical characterization under dry and wet conditions showed that increasing collagen content resulted in a tremendous decrease in the mechanical properties. However, crosslinking resulted in the increase in elastic modulus from 47 MPa to 83 MPa for the wet PLGA/Collagen 80/20 blend system, with little effect on the tensile strength. In conclusion, the aligned nanofibrous scaffold used in this study constitutes a promising material for bone tissue engineering.     

Fabrication of fibrous poly (ɛ-caprolactone) nano-fibers containing cerium doped-bioglasses nanoparticles encapsulated collagen

Novel nanosized designed ceramic powders, cerium (Ce) doped bioglass (BG) with various doped Ce content, were synthesized by sol–gel method in order to be employed in the development of PCL fibrous scaffold for bone tissue engineering applications. Characterization techniques such as X-ray diffraction analysis, transmission electron microscopy, Fourier transform infrared spectroscopy, and energy-dispersive X-ray spectroscopy were employed to evaluate the developed Ce doped BG powders. The results confirmed successful doping of Ce inside BG structure. 0, 1, 3, and 10 wt% Ce doped 58S BG were successfully encapsulated in the collagen microspheres by water-in-oil emulsion method and the average particle size and hydrodynamic diameter of microspheres were determined using scanning electron microscopy and dynamic light scattering analysis, respectively. Next, 0, 1, 3, and 10 wt% Ce doped 58S BG encapsulated collagen microspheres were loaded inside the Poly(ɛ-caprolactone) fibrous scaffold and their in vitro bioactivity and biocompatibility properties were evaluated. The results of soaking samples in the simulated body fluid showed that all Ce doped 58S BG encapsulated collagen microspheres loaded PCL fibrous scaffold have acceptable bioactivity and apatite formation ability over time. The biocompatibility evaluation of developed scaffolds showed high viability and proliferation of MG63 cells cultured on the surface of 3% Ce doped 58S BG encapsulated collagen microsphere loaded in the PCL fibrous scaffold and its high potential ability for bone tissue engineering applications. These results potentially open new aspects for scaffolds aimed at the regeneration of bone defects.     

Graphene Oxide Framework Structures and Coatings: Impact on Cell Adhesion and Pre-Vascularization Processes for Bone Grafts

Graphene oxide (GO) is a promising material for bone tissue engineering, but the validation of its molecular biological effects, especially in the context of clinically applied materials, is still limited. In this study, we compare the effects of graphene oxide framework structures (F-GO) and reduced graphene oxide-based framework structures (F-rGO) as scaffold material with a special focus on vascularization associated processes and mechanisms in the bone. Highly porous networks of zinc oxide tetrapods serving as sacrificial templates were used to create F-GO and F-rGO with porosities >99% consisting of hollow interconnected microtubes. Framework materials were seeded with human mesenchymal stem cells (MSC), and the cell response was evaluated by confocal laser scanning microscopy (CLSM), deoxyribonucleic acid (DNA) quantification, real-time polymerase chain reaction (RT-PCR), enzyme-linked immunosorbent assay (ELISA), and alkaline phosphatase activity (ALP) to define their impact on cellular adhesion, osteogenic differentiation, and secretion of vascular growth factors. F-GO based scaffolds improved adhesion and growth of MSC as indicated by CLSM and DNA quantification. Further, F-GO showed a better vascular endothelial growth factor (VEGF) binding capacity and improved cell growth as well as the formation of microvascular capillary-like structures in co-cultures with outgrowth endothelial cells (OEC). These results clearly favored non-reduced graphene oxide in the form of F-GO for bone regeneration applications. To study GO in the context of a clinically used implant material, we coated a commercially available xenograft (Bio-Oss^® block) with GO and compared the growth of MSC in monoculture and in coculture with OEC to the native scaffold. We observed a significantly improved growth of MSC and formation of prevascular structures on coated Bio-Oss^®, again associated with a higher VEGF binding capacity. We conclude that graphene oxide coating of this clinically used, but highly debiologized bone graft improves MSC cell adhesion and vascularization.     

Electrospinning of poly (3-hydroxybutyric acid) and gelatin blended thin films: fabrication, characterization, and application in skin regeneration

A tissue engineering scaffold should mimic the structure and biological function of native extracellular matrix proteins. Electrospinning is a simple and versatile method to produce ultrathin fibers for tissue engineering. Blended submicron fibers of poly (3-hydroxybutyric acid) and gelatin were electrospun using 1,1,1,3,3,3 hexafluoro-2-propanol as solvent. Cross linking of fibers was achieved using glutaraldehyde, and the resultant fibers were tested and analyzed using scanning electron microscopy (SEM), differential scanning calorimetry, thermogravimetric analysis, X-ray diffraction, and Fourier transformed infrared spectroscopy (FTIR).The fibers were found to exhibit good tensile strength. Degradation studies were performed and analyzed using SEM and FTIR and proved the stability of fibers for tissue engineering applications. The fibrous scaffold supported the growth and rapid proliferation of human dermal fibroblasts and keratinocytes with normal morphology, thus proving its reliability in using it as a potential scaffold for skin regeneration.