A Biomimetic Collagen-Apatite Scaffold with a Multi-Level Lamellar Structure for Bone Tissue Engineering.
ABSTRACT: Collagen-apatite (Col-Ap) scaffolds have been widely employed for bone tissue engineering. We fabricated a Col-Ap scaffold with a unique multi-level lamellar structure consisting of co-aligned micro and macro pores. The basic building blocks of this scaffold are bone-like mineralized collagen fibers developed via a biomimetic self-assembly process in a collagen-containing modified simulated body fluid (m-SBF). This biomimetic method preserves the structural integrity and great tensile strength of collagen by reinforcing the collagen hydrogel with apatite nano-particles. Unidirectional aligned macro pores with a size of 63.8 to 344 ?m are created by controlling the freezing rate and direction. The thickness of Col-Ap lamellae can be adjusted in the range 3.6 to 23 ?m depending on the self-compression time. Furthermore, the multi-level lamellar structure has led to a twelve-fold increase in Young's modulus and a two-fold increase in the compression modulus along the aligned direction compared to a scaffold of the same composition with an isotropic equiaxed pore structure. Moreover, this novel lamellar scaffold supports the attachment and spreading of MC3T3-E1osteoblasts. Therefore, owing to the biomimetic composition, tunable structure, improved mechanical strength, and good biocompatibility of this novel scaffold, it has great potential to be used in bone tissue engineering applications.
Project description:Various kinds of three-dimensional (3D) scaffolds have been designed to mimic the biological spontaneous bone formation characteristics by providing a suitable microenvironment for osteogenesis. In view of this, a natural bone-liked composite scaffold, which was combined with inorganic (hydroxyapatite, Hap) and organic (type I collagen, Col) phases, has been developed through a self-assembly process. This 3D porous scaffold consisting of a c-axis of Hap nanocrystals (nHap) aligning along Col fibrils arrangement is similar to natural bone architecture. A significant increase in mechanical strength and elastic modulus of nHap/Col scaffold is achieved through biomimetic mineralization process when compared with simple mixture of collagen and hydroxyapatite method. It is suggested that the self-organization of Hap and Col produced in vivo could also be achieved in vitro. The oriented nHap/Col composite not only possesses bone-like microstructure and adequate mechanical properties but also enhances the regeneration and reorganization abilities of bone tissue. These results demonstrated that biomimetic nHap/Col can be successfully reconstructed as a bone graft substitute in bone tissue engineering.
Project description:The hierarchical structure of bone and intrinsic material properties of its two primary constituents, carbonated apatite and fibrillar collagen, when being synergistically organized into an interpenetrating hard-soft composite, contribute to its excellent mechanical properties. Lamellar bone is the predominant structural motif in mammalian hard tissues; therefore, we believe the fabrication of a collagen/apatite composite with a hierarchical structure that emulates bone, consisting of a dense lamellar microstructure and a mineralized collagen fibril nanostructure, is an important first step toward the goal of regenerative bone tissue engineering. In this work, we exploit the liquid crystalline properties of collagen to fabricate dense matrices that assemble with cholesteric organization. The matrices were crosslinked via carbodiimide chemistry to improve mechanical properties, and are subsequently mineralized via the polymer-induced liquid-precursor (PILP) process to promote intrafibrillar mineralization. Neither the crosslinking procedure nor the mineralization affected the cholesteric collagen microstructures; notably, there was a positive trend toward higher stiffness with increasing crosslink density when measured by cantilever-based atomic force microscopy (AFM) nanoindentation. In the dry state, the average moduli of moderately (X51; 4.8 ± 4.3 GPa) and highly (X76; 7.8 ± 6.7 GPa) crosslinked PILP-mineralized liquid crystalline collagen (LCC) scaffolds were higher than the average modulus of bovine bone (5.5 ± 5.6 GPa).
Project description:Rotator cuff tears are common and cause a great deal of lost productivity, pain, and disability. Tears are typically repaired by suturing the tendon back to its bony attachment. Unfortunately, the structural (e.g., aligned collagen) and compositional (e.g., a gradient in mineral) elements that produce a robust attachment in the healthy tissue are not regenerated during healing, and the repair is prone to failure. Two features of the failed healing response are deposition of poorly aligned scar tissue and loss of bone at the repair site. Therefore, the objective of the current study was to improve tendon-to-bone healing by promoting aligned collagen deposition and increased bone formation using a biomimetic scaffold seeded with pluripotent cells. An aligned nanofibrous poly(lactic-co-glycolic acid) scaffold with a gradient in mineral content was seeded with adipose-derived stromal cells (ASCs) and implanted at the repair site of a rat rotator cuff model. In one group, cells were transduced with the osteogenic factor bone morphogenetic protein 2 (BMP2). The healing response was examined in four groups (suture only, acellular scaffold, cellular scaffold, and cellular BMP2 scaffold) using histologic, bone morphology, and biomechanical outcomes at 14, 28, and 56 days. Histologically, the healing interface was dominated by a fibrovascular scar response in all groups. The acellular scaffold group showed a delayed healing response compared to the other groups. When examining bone morphology parameters, bone loss was evident in the cellular BMP2 group compared to other groups at 28 days. When examining repair-site mechanical properties, strength and modulus were decreased in the cellular BMP2 groups compared to other groups at 28 and 56 days. These results indicated that tendon-to-bone healing in this animal model was dominated by scar formation, preventing any positive effects of the implanted biomimetic scaffold. Furthermore, cells transduced with the osteogenic factor BMP2 led to impaired healing, suggesting that this growth factor should not be used in the tendon-to-bone repair setting.
Project description:Biominerals exhibit complex hierarchical structures derived from bottom-up self-assembly mechanisms. Type I collagen serves as the building block for mineralized tissues such as bone and dentin. In the present study, 250-300 ?m thick, partially demineralized collagen scaffolds exhibiting a gradient of demineralization from the base to surface were mineralized using a classical top-down approach and a non-classical bottom-up approach. The top-down approach involved epitaxial growth over seed crystallites. The bottom-up approach utilized biomimetic analogs of matrix proteins to stabilize amorphous calcium phosphate nanoprecursors and template apatite nucleation and growth within the collagen matrix. Micro-computed tomography and transmission electron microscopy were employed to examine mineral uptake and apatite arrangement within the mineralized collagen matrix. The top-down approach could mineralize only the base of the partially demineralized scaffold, where remnant seed crystallites were abundant. Minimal mineralization was observed along the surface of the scaffold; extrafibrillar mineralization was predominantly observed. Conversely, the entire partially demineralized scaffold, including apatite-depleted collagen fibrils, was mineralized by the bottom-up approach, with evidence of both intrafibrillar and extrafibrillar mineralization. Understanding the different mechanisms involved in these two mineralization approaches is pivotal in adopting the optimum strategy for fabricating novel nanostructured materials in bioengineering research.
Project description:Neural tissue regeneration is a significant challenge, because severe nerve injury is quite difficult to regenerate spontaneously. Although, many studies have been devoted to promote nerve regeneration, there are still many technical challenges to achieve satisfactory results. In this study, we designed biomimetic matrices composed of aligned laminin core-polydioxanone/collagen shell (Lam-PDO/Col) fibers, which can provide both topographical and biochemical cues for promoting neuritogenesis. The aligned Lam-PDO/Col core-shell fiber matrices were fabricated by magnetic field-assisted electrospinning with the coaxial system, and their potential as biofunctional scaffolds for promoting neuritogenesis was explored. It was demonstrated that the aligned Lam-PDO/Col core-shell fibers were successfully fabricated, and the laminin in the core of fibers was steadily and continuously released from fibers. In addition, the cellular behaviors of hippocampal neuronal cells on the matrices were significantly enhanced. Moreover, the aligned Lam-PDO/Col fiber matrices effectively improved and guided neurite outgrowth as well as the neurogenic differentiation by providing both topographical and biochemical cues through aligned fiber structure and sustained release of laminin. Collectively, it is suggested that the aligned Lam-PDO/Col core-shell fiber matrices are one of the most promising approaches for promoting neuritogenesis and neural tissue regeneration.
Project description:Muscle regeneration can be permanently impaired by traumatic injuries, despite the high regenerative capacity of skeletal muscle. Implantation of engineered biomimetic scaffolds to the site of muscle ablation may serve as an attractive off-the-shelf therapeutic approach. The objective of the study was to histologically assess the therapeutic benefit of a three-dimensional spatially patterned collagen scaffold, in conjunction with rehabilitative exercise, for treatment of volumetric muscle loss. To mimic the physiologic organization of skeletal muscle, which is generally composed of myofibers aligned in parallel, three-dimensional parallel-aligned nanofibrillar collagen scaffolds were fabricated. When implanted into the ablated murine tibialis anterior muscle, the aligned nanofibrillar scaffolds, in conjunction with voluntary caged wheel exercise, significantly improved the density of perfused microvessels, in comparison to treatments of the randomly oriented nanofibrillar scaffold, decellularized scaffold, or in the untreated control group. The abundance of neuromuscular junctions was 19-fold higher when treated with aligned nanofibrillar scaffolds in conjunction with exercise, in comparison to treatment of aligned scaffold without exercise. Although, the density of de novo myofibers was not significantly improved by aligned scaffolds, regardless of exercise activity, the cross-sectional area of regenerating myofibers was increased by?>?60% when treated with either aligned and randomly oriented scaffolds, in comparison to treatment of decellularized scaffold or untreated controls. These findings demonstrate that voluntary exercise improved the regenerative effect of aligned scaffolds by augmenting neurovascularization, and have important implications in the design of engineered biomimetic scaffolds for treatment of traumatic muscle injury.
Project description:Recapitulation of the form and function of complex tissue organization using appropriate biomaterials impacts success in tissue engineering endeavors. The annulus fibrosus (AF) represents a complex, multilamellar, hierarchical structure consisting of collagen, proteoglycans, and elastic fibers. To mimic the intricacy of AF anatomy, a silk protein-based multilayered, disc-like angle-ply construct was fabricated, consisting of concentric layers of lamellar sheets. Scanning electron microscopy and fluorescence image analysis revealed cross-aligned and lamellar characteristics of the construct, mimicking the native hierarchical architecture of the AF. Induction of secondary structure in the silk constructs was confirmed by infrared spectroscopy and X-ray diffraction. The constructs showed a compressive modulus of 499.18 ± 86.45 kPa. Constructs seeded with porcine AF cells and human mesenchymal stem cells (hMSCs) showed ?2.2-fold and ?1.7-fold increases in proliferation on day 14, respectively, compared with initial seeding. Biochemical analysis, histology, and immunohistochemistry results showed the deposition of AF-specific extracellular matrix (sulfated glycosaminoglycan and collagen type I), indicating a favorable environment for both cell types, which was further validated by the expression of AF tissue-specific genes. The constructs seeded with porcine AF cells showed ?11-, ?5.1-, and ?6.7-fold increases in col I? 1, sox 9, and aggrecan genes, respectively. The differentiation of hMSCs to AF-like tissue was evident from the enhanced expression of the AF-specific genes. Overall, the constructs supported cell proliferation, differentiation, and ECM deposition resulting in AF-like tissue features based on ECM deposition and morphology, indicating potential for future studies related to intervertebral disc replacement therapy.
Project description:Fiber bundles are present in many tissues throughout the body. In most cases, collagen subunits spontaneously self-assemble into a fibrilar structure that provides ductility to bone and constitutes the basis of muscle contraction. Translating these natural architectural features into a biomimetic scaffold still remains a great challenge. Here, we propose a simple strategy to engineer biomimetic fiber bundles that replicate the self-assembly and hierarchy of natural collagen fibers. The electrostatic interaction of methacrylated gellan gum (MeGG) with a countercharged chitosan (CHT) polymer led to the complexation of the polyelectrolytes. When directed through a polydimethylsiloxane (PDMS) channel, the polyelectrolytes formed a hierarchical fibrous hydrogel demonstrating nano-scale periodic light/dark bands similar to D-periodic bands in native collagen and aligned parallel fibrils at micro-scale. Importantly, collagen-mimicking hydrogel fibers exhibited robust mechanical properties (MPa scale) at a single fiber bundle level and enabled encapsulation of cells inside the fibers under cell-friendly mild conditions. Presence of carboxyl- (in gellan gum) or amino- (in chitosan) functionalities further enabled controlled peptide functionalization such as RGD for biochemical mimicry (cell adhesion sites) of native collagen. This biomimetic aligned fibrous hydrogel system can potentially be used as a scaffold for tissue engineering as well as a drug/gene delivery vehicle.
Project description:Hamstring tendon autograft is a routine graft for anterior cruciate ligament (ACL) reconstruction. However, ways of improving the healing between the tendon and bone is often overlooked in clinical practice. This issue can be addressed by using a biomimetic scaffold. Herein, a biomimetic nanofiber membrane of polycaprolactone/nanohydroxyapatite/collagen (PCL/nHAp/Col) is fabricated that mimics the composition of native bone tissue for promoting tendon-bone healing. This membrane has good cytocompatibility, allowing for osteoblast cell adhesion and growth and bone formation. As a result, MC3T3 cells reveal a higher mineralization level in PCL/nHAp/Col membrane compared with PCL membrane alone. Further in vivo studies in ACL reconstruction in a rabbit model shows that PCL/nHAp/Col-wrapped tendon may afford superior tissue integration to nonwrapped tendon in the interface between the tendon and host bone as well as improved mechanical strength. This study shows that PCL/nHAp/Col nanofiber membrane wrapping of autologous tendon is effective for improving tendon healing with host bone in ACL reconstruction.
Project description:Properties of the cell-material interface are determining factors in the successful function of cells for cartilage tissue engineering. Currently, cell adhesion is commonly promoted through the use of polypeptides; however, due to their lack of complementary or modulatory domains, polypeptides must be modified to improve their ability to promote adhesion. In this study, we utilized the principle of matrix-based biomimetic modification and a recombinant protein, which spans fragments 7-10 of fibronectin module III (heterophilic motif) and extracellular domains 1-2 of cadherin-11 (rFN/Cad-11) (homophilic motif), to modify the interface of collagen type II (Col II) sponges. We showed that the designed material was able to stimulate cell proliferation and promote better chondrogenic differentiation of rabbit mesenchymal stem cells (MSCs) in vitro than both the FN modified surfaces and the negative control. Further, the Col II/rFN/Cad-11-MSCs composite stimulated cartilage formation in vivo; the chondrogenic effect of Col II alone was much less significant. These results suggested that the rFN/Cad-11-modified collagen type II biomimetic interface has dual biological functions of promoting adhesion and stimulating chondrogenic differentiation. This substance, thus, may serve as an ideal scaffold material for cartilage tissue engineering, enhancing repair of injured cartilage in vivo.