Systematic studies of a self-assembling peptide nanofiber scaffold with other scaffolds

A designer self-assembling peptide nanofiber scaffold has been systematically studied with 10 commonly used scaffolds in a several week study using neural stem cells (NSC), a potential therapeutic source for cellular transplantations in nervous system injuries. These cells not only provide a good in vitro model for the development and regeneration of the nervous system, but may also be helpful in testing for cytotoxicity, cellular adhesion, and differentiation properties of biological and synthetic scaffolds used in medical practices. We tested the self-assembling peptide nanofiber scaffold with the most commonly used scaffolds for tissue engineering and regenerative medicine including PLLA, PLGA, PCLA, collagen I, collagen IV, and Matrigel. Additionally, each scaffold was coated with laminin in order to evaluate the utility of this surface treatment. Each scaffold was evaluated by measuring cell viability, differentiation and maturation of the differentiated stem cell progeny (i.e. progenitor cells, astrocytes, oligodendrocytes, and neurons) over 4 weeks. The optimal scaffold should show high numbers of living and differentiated cells. In addition, it was demonstrated that the laminin surface treatment is capable of improving the overall scaffold performance. The designer self-assembling peptide RADA16 nanofiber scaffold represents a new class of biologically inspired material. The well-defined molecular structure with considerable potential for further functionalization and slow drug delivery makes the designer peptide scaffolds a very attractive class of biological material for a number of applications. The peptide nanofiber scaffold is comparable with the clinically approved synthetic scaffolds. The peptide scaffolds are not only pure, but also have the potential to be further designed at the molecular level, thus they promise to be useful for cell adhesion and differentiation studies as well as for future biomedical and clinical studies.     

Designer self-assembling peptide scaffold stimulates pre-osteoblast attachment, spreading and proliferation

A new peptide scaffold was made by mixing pure RADA16 (Ac-RADARADARADARADA-CONH2) and designer peptide RGDA16 (Ac-RADARGDARADARGDA-CONH2) solutions, and investigate any effect on attachment, spreading and proliferation of pre-osteoblast (MC3T3-E1). The peptides, RADA16 and RGDA16, were custom-synthesized. They were solubilized in deionized water at a concentration of 10 mg/ml (1% w/v), the RGDA16 peptide solution was mixed 1:1 with RADA16 solution and a new peptide solution RGDAmix was produced. The RGDAmix and RADA16 solution were directly loaded in 96-well plates and cover slips, and two different peptide scaffolds were formed with the addition of maintenance medium (α-MEM) in several minutes. About 1.0 × 10⁴ MC3T3-E1 cells were seeded on each hydrogel scaffold, and then the cell morphological changes were observed using a fluorescence microscope at 1 h, 3 h and 24 h timepoint, respectively. Cell attachment was evaluated 1 h, 3 h and 24 h after cell seeding and cell proliferation was determined 4d, 7d and 14d after cell seeding. The RGDAmix scaffold significantly promoted the initial cell attachment compared with the RADA16 scaffold. MC3T3-E1 cells adhered and spread well on both scaffolds, however, cells spread better on the RGDAmix scaffold than on the RADA16 scaffold. Cell proliferation was greatly stimulated when cultured on RGDAmix scaffold. The RGD sequence contained peptide scaffold RGDAmix significantly enhances MC3T3-E1 cells attachment, spreading and proliferation.     

A novel honeycomb cell assay kit designed for evaluating horizontal cell migration in response to functionalized self-assembling peptide hydrogels

A clear understanding on cell migration behaviors contributes to designing novel biomaterials in tissue engineering and elucidating related tissue regeneration processes. Many traditional evaluation methods on cell migration including scratch assay and transwell migration assay possess all kinds of limitations. In this study, a novel honeycomb cell assay kit was designed and made of photosensitive resin by 3D printing. This kit has seven hexagonal culture chambers so that it can evaluate the horizontal cell migration behavior in response to six surrounding environments simultaneously, eliminating the effect of gravity on cells. Here this cell assay kit was successfully applied to evaluate endothelial cell migration cultured on self-assembling peptide (SAP) RADA (AcN-RADARADARADARADA-CONH₂) nanofiber hydrogel toward different functionalized SAP hydrogels. Our results indicated that the functionalized RADA hydrogels with different concentration of bioactive motifs of KLT or PRG could induce cell migration in a dose-dependent manner. The total number and migration distance of endothelial cells on functionalized SAP hydrogels significantly increased with increasing concentration of bioactive motif PRG or KLT. Therefore, the honeycomb cell assay kit provides a simple, efficient and convenient tool to investigate cell migration behavior in response to multi-environments simultaneously.     

Growth of fibroblast and vascular smooth muscle cells in fibroin/collagen scaffold

In our recent study, a novel fibroin/collagen scaffold with improved mechanical properties and controllable porous structure was prepared through freeze–drying method. In this research, the cyto-compatibility was further studied, using fibroblast and vascular smooth muscle cells (VSMC) as the model cells. MTT results indicated that the growth of fibroblast and VSMC both further improved in the fibroin/collagen scaffold than in pure fibroin scaffolds. The confocal and SEM results showed that fibroblast cells and VSMCs had better adhesion and spreading properties in the fibroin/collagen scaffolds. Although further studies, such as the extracellular matrix production and the functional gene expression, are necessary to clarify the biocompatibility of the fibroin/collagen scaffolds, the present results indicate that the fibroin/collagen scaffold is a new scaffold material suitable for tissue engineering. On the other hand, the mild and all-aqueous preparation processes also make it possible to embed different growth factors inside the scaffolds to maximize cell functions and the formation of specific tissues.     

复合材料梯度结构组织工程支架建模方法

使用传统方法制造组织工程细胞载体支架存在材料单一，结构简单的缺点，提出了一种基于快速成形技术（MEM工艺，类似于FDM）的复合材料梯度结构组织工程支架的建模方法，使用这种方法，可以按照支架的特点和要求，分别采用结构模板和材料模板进行支架的结构设计和材料设计，从而实现支架的多种材料和梯度结构。     

Electrospun PCL/PLA/HA based nanofibers as scaffold for osteoblast-like cells

Polycaprolactone (PCL), poly (lactic acid) (PLA) and hydroxyapatite (HA) are frequently used as materials for tissue engineering. In this study, PCL/PLA/HA nanofiber mats with different weight ratio were prepared using electrospinning. Their structure and morphology were studied by FTIR and FESEM. FTIR results demonstrated that the HA particles were successfully incorporated into the PCL/PLA nanofibers. The FESEM images showed that the surface of fibers became coarser with the introduction of HA nanoparticles into PCL/PLA system. Furthermore, the addition of HA led to the decreasing of fiber diameter. The average diameters of PCL/PLA/HA nanofiber were in the range of 300-600 nm, while that of PCL/PLA was 776 +/- 15.4 nm. The effect of nanofiber composition on the osteoblast-like MC3T3-E1 cell adhesion and proliferation were investigated as the preliminary biological evaluation of the scaffold. The MC3T3-E1 cell could be attached actively on all the scaffolds. The MTT assay revealed that PCL/PLA/HA scaffold shows significantly higher cell proliferation than PCL/PLA scaffolds. After 15 days of culture, mineral particles on the surface of the cells was appeared on PCL/PLA/HA nanofibers while normal cell spreading morphology on PCL/PLA nanofibers. These results manifested that electrospun PCL/PLA/HA scaffolds could enhance bone regeneration, showing their marvelous prospect as scaffolds for bone tissue engineering.     

Preparation and characterization of polycaprolactone/forsterite nanocomposite porous scaffolds designed for bone tissue regeneration

Biocomposite scaffolds made from polymers and bioceramics can provide the mechanical structure necessary for osteoinductivity in the growth of new bone. The aim of this research was to investigate the properties of a novel nanocomposite scaffold made from a combination of polycaprolactone (PCL) and forsterite nanopowder which could find use in bone tissue engineering applications. The scaffold itself was fabricated by a method of solvent casting and particle leaching. The effect of forsterite content on the mechanical properties, bioactivity, biodegradability, and cytotoxicity of the scaffolds was investigated. Significant improvement in the mechanical properties was observed in the nanocomposite scaffolds as compared to that seen in the pure PCL scaffolds. Bioactivity was also observed in the nanocomposite scaffolds, a trait which was not present in the pure PCL scaffolds. Biodegradation assay indicated that the addition of forsterite nanopowder could modulate the degradation rate of PCL. In vitro tests of cytotoxicity and osteoblast proliferation showed that the nanocomposite scaffolds were non-cytotoxic, thereby allowing cells to adhere, grow, and proliferate on the surface of these scaffolds. The results obtained in this experiment suggest that the combination of PCL with forsterite nanopowder can be used to form scaffolds suitable for use in bone tissue engineering. The exact material behavior required can be adjusted through variation of the ratio between PCL and forsterite nanopowder used to form the scaffold.     

PLLA–collagen and PLLA–gelatin hybrid scaffolds with funnel-like porous structure for skin tissue engineering

In skin tissue engineering, a three-dimensional porous scaffold is necessary to support cell adhesion and proliferation and to guide cells moving into the repair area in the wound healing process. Structurally, the porous scaffold should have an open and interconnected porous architecture to facilitate homogenous cell distribution. Moreover, the scaffolds should be mechanically strong to protect deformation during the formation of new skin. In this study, the hybrid scaffolds were prepared by forming funnel-like collagen or gelatin sponge on a woven poly(l-lactic acid) (PLLA) mesh. The hybrid scaffolds combined the advantages of both collagen or gelatin (good cell-interactions) and PLLA mesh (high mechanical strength). The hybrid scaffolds were used to culture dermal fibroblasts for dermal tissue engineering. The funnel-like porous structure promoted homogeneous cell distribution and extracellular matrix production. The PLLA mesh reinforced the scaffold to avoid deformation. Subcutaneous implantation showed that the PLLA–collagen and PLLA–gelatin scaffolds promoted the regeneration of dermal tissue and epidermis and reduced contraction during the formation of new tissue. These results indicate that funnel-like hybrid scaffolds can be used for skin tissue regeneration.     

Fabrication and characterization of <Emphasis Type="Italic">Antheraea pernyi</Emphasis> silk fibroin-blended P(LLA-CL) nanofibrous scaffolds for peripheral nerve tissue engineering

Electrospun nanofibers have gained widespreading interest for tissue engineering application. In the present study, ApF/P(LLA-CL) nanofibrous scaffolds were fabricated via electrospinning. The feasibility of the material as tissue engineering nerve scaffold was investigated in vitro. The average diameter increased with decreasing the blend ratio of ApF to P(LLA-CL). Characterization of ¹³C NMR and FTIR clarified that there is no obvious chemical bond reaction between ApF and P(LLA-CL). The tensile strength and elongation at break increased with the content increase of P(LLA-CL). The surface hydrophilic property of nanofibrous scaffolds enhanced with the increased content of ApF. Cell viability studies with Schwann cells demonstrated that ApF/P(LLA-CL) blended nanofibrous scaffolds significantly promoted cell growth as compare to P(LLA-CL), especially when the weight ratio of ApF to P(LLA-CL) was 25:75. The present work provides a basis for further studies of this novel nanofibrous material (ApF/P(LLA-CL)) in peripheral nerve tissue repair or regeneration.     

The influence of fiber thickness, wall thickness and gap distance on the spiral nanofibrous scaffolds for bone tissue engineering

We have developed a 3D nanofibrous spiral scaffold for bone tissue engineering which has shown enhanced cell attachment, proliferation and differentiation compared to traditional cylindrical scaffolds due to the spiral structures and the nanofiber incorporation. Some important parameters of these spiral scaffolds including gap distance, wall thickness and especially fiber thickness are crucial to the performance of the spiral structured scaffolds. In this study, we investigated the fiber thickness, gap distance and wall thickness of the spiral structure on the behavior of osteoblast cells. The human osteoblast cells are seeded on spiral structured scaffolds with various fiber thickness, gap distance and wall thickness and cell attachment, proliferation, differentiation and mineralized matrix deposition on the scaffolds are evaluated. It was found that increasing the thickness of nanofiber layer not only limited the cell infiltration into the scaffolds, but also restrained the osteoblastic cell phenotype development. Moreover, the geometric effect studies indicated that scaffolds with the thinner wall and gap distance 0.2 mm show the best bioactivity for osteoblasts.     

Development of a novel collagen–GAG nanofibrous scaffold via electrospinning

Collagen and glycosaminoglycan (GAG) are native constituents of human tissues and are widely utilized to fabricate scaffolds serving as an analog of native extracellular matrix (ECM).The development of blended collagen and GAG scaffolds may potentially be used in many soft tissue engineering applications since the scaffolds mimic the structure and biological function of native ECM. In this study, we were able to obtain a novel nanofibrous collagen–GAG scaffold by electrospinning with collagen and chondroitin sulfate (CS), a widely used GAG. The electrospun collagen–GAG scaffold exhibited a uniform fiber structure in nano-scale diameter. By crosslinking with glutaraldehyde vapor, the collagen–GAG scaffolds could resist from collagenase degradation and enhance the biostability of the scaffolds. This led to the increased proliferation of rabbit conjunctiva fibroblast on the scaffolds. Incorporation of CS into collagen nanofibers without crosslinking did not increase the biostability but still promoted cell growth. In conclusion, the electrospun collagen–GAG scaffolds, with high surface-to-volume ratio, may potentially provide a better environment for tissue formation/biosynthesis compared with the traditional scaffolds.     

Poly(L-lactic acid) nanocylinders as nanofibrous structures for macroporous gelatin scaffolds

Electrospun Nanofiber sheets have been shown to mimic the structure of extracellular matrix (ECM). Although these nanofibers have shown great potential for use as tissue engineering scaffolds, it is difficult for the electrospun nanofiber based sheets to be shaped into the desired three-dimensional structure. In this study, poly(L-lactic acid) (PLLA), a biodegradable and biocompatible polyester, was electrospun to produce nanofibers that were treated with an amino group containing base in order to fabricate polymeric nanocylinders. The aspect ratio of the PLLA nanocylinders was tunable by varying the aminolysis time and density of the amino group containing base. The effects of changes in nanofibrous morphology of the PLLA nanocylinders/macro-porous gelatin scaffolds on cell adhesion and proliferation were evaluated. The results revealed different cell morphology, adhesion, and proliferation in the nanocylinders composite gelatin scaffold versus gelatin scaffold alone. Confocal laser scanning microscopy observation showed more spreading and a more flattened cell morphology after NIH3T3 cells were cultured on PLLA nanocylinders/gelatin scaffolds for 10 hours and 4 days. These results indicate that the gelatin/PLLA nanocylinder composite is a promising way to fabricate 3D nanofibrous scaffolds that accelerates cell adhesion and proliferation for tissue engineering.     

Electrospun poly(vinylidene fluoride-trifluoroethylene)/zinc oxide nanocomposite tissue engineering scaffolds with enhanced cell adhesion and blood vessel formation

Piezoelectric materials that generate electrical signals in response to mechanical strain can be used in tissue engineering to stimulate cell proliferation.Poly (vinylidene fluoride-trifluoroethylene) (P(VDF-TrFE)),a piezoelectric polymer,is widely used in biomaterial applications.We hypothesized that incorporation of zinc oxide (ZnO) nanoparticles into the P(VDF-TrFE) matrix could promote adhesion,migration,and proliferation of cells,as well as blood vessel formation (angiogenesis).In this study,we fabricated and comprehensively characterized a novel electrospun P(VDF-TrFE)/ZnO nanocomposite tissue engineering scaffold.We analyzed the morphological features of the polymeric matrix by scanning electron microscopy,and utilized Fourier transform infrared spectroscopy,X-ray diffraction,and differential scanning calorimetry to examine changes in the crystalline phases of the copolymer due to addition of the nanoparticles.We detected no or minimal adverse effects of the biomaterials with regard to blood compatibility in vitro,biocompatibility,and cytotoxicity,indicating that P(VDF-TrFE)/ZnO nanocomposite scaffolds are suitable for tissue engineering applications.Interestingly,human mesenchymal stem cells (hMSCs) and human umbilical vein endothelial cells cultured on the nanocomposite scaffolds exhibited higher cell viability,adhesion,and proliferation compared to cells cultured on tissue culture plates or neat P(VDF-TrFE) scaffolds.Nanocomposite scaffolds implanted into rats with or without hMSCs did not elicit immunological responses,as assessed by macroscopic analysis and histology.Importantly,nanocomposite scaffolds promoted angiogenesis,which was increased in scaffolds pre-seeded with hMSCs.Overall,our results highlight the potential of these novel P(VDF-TrFE)/ZnO nanocomposites for use in tissue engineering,due to their biocompatibility and ability to promote cell adhesion and angiogenesis.     

PLLA–collagen and PLLA–gelatin hybrid scaffolds with funnel-like porous structure for skin tissue engineering

Abstract

In skin tissue engineering, a three-dimensional porous scaffold is necessary to support cell adhesion and proliferation and to guide cells moving into the repair area in the wound healing process. Structurally, the porous scaffold should have an open and interconnected porous architecture to facilitate homogenous cell distribution. Moreover, the scaffolds should be mechanically strong to protect deformation during the formation of new skin. In this study, the hybrid scaffolds were prepared by forming funnel-like collagen or gelatin sponge on a woven poly(l-lactic acid) (PLLA) mesh. The hybrid scaffolds combined the advantages of both collagen or gelatin (good cell-interactions) and PLLA mesh (high mechanical strength). The hybrid scaffolds were used to culture dermal fibroblasts for dermal tissue engineering. The funnel-like porous structure promoted homogeneous cell distribution and extracellular matrix production. The PLLA mesh reinforced the scaffold to avoid deformation. Subcutaneous implantation showed that the PLLA–collagen and PLLA–gelatin scaffolds promoted the regeneration of dermal tissue and epidermis and reduced contraction during the formation of new tissue. These results indicate that funnel-like hybrid scaffolds can be used for skin tissue regeneration.     

In vitro investigations of a novel wound dressing concept based on biodegradable polyurethane

Non-healing and partially healing wounds are an important problem not only for the patient but also for the public health care system. Current treatment solutions are far from optimal regarding the chosen material properties as well as price and source. Biodegradable polyurethane (PUR) scaffolds have shown great promise for in vivo tissue engineering approaches, but accomplishment of the goal of scaffold degradation and new tissue formation developing in parallel has not been observed so far in skin wound repair. In this study, the mechanical properties and degradation behavior as well as the biocompatibility of a low-cost synthetic, pathogen-free, biocompatible and biodegradable extracellular matrix mimicking a PUR scaffold was evaluated in vitro. The novel PUR scaffolds were found to meet all the requirements for optimal scaffolds and wound dressings. These three-dimensional scaffolds are soft, highly porous, and form-stable and can be easily cut into any shape desired. All the material formulations investigated were found to be nontoxic. One formulation was able to be defined that supported both good fibroblast cell attachment and cell proliferation to colonize the scaffold. Tunable biodegradation velocity of the materials could be observed, and the results additionally indicated that calcium plays a crucial role in PUR degradation. Our results suggest that the PUR materials evaluated in this study are promising candidates for next-generation wound treatment systems and support the concept of using foam scaffolds for improved in vivo tissue engineering and regeneration.     

Fabrication of Nanofiber Reinforced Protein Structures For Tissue Engineering

Extracellular matrices and degradable nanofibers are two very promising materials in the field of tissue engineering; however both of these structures face limitations as tissue engineering scaffolds. Extracellular matrices, such as collagen, gelatin, and laminin, have excellent biocompatibility and allow cell in growth and survival, but structural weakness makes them difficult to handle and greatly limits their uses. Degradable nanofibers support cell attachment and can provide structural support and directional guidance, but individual degradable nanofibers are fragile and have a tendency to form dense fiber bundles which limit cell penetration into the spaces between the nanofibers, especially in the case of aligned nanofibers. To overcome these difficulties, degradable loose nanofibers were embedded in protein matrix in an attempt to fabricate a hybrid scaffold with improved properties, such as improved strength, guidance, spacing among nanofibers, etc. Polycaprolactone (PCL) was used as a model material for degradable nanofibers. Gelatin was employed as a model protein for matrix structure formation. Thin hybrid films (average thickness = 2.78 um) were fabricated by wetting the loose aligned undirectional nanofiber arrays or loose aligned bi-directional nanofiber grids with a gelatin aqueous solution, which also allows for live cell loading into the nanofiber-protein composite if cell are premixed with protein solution or on the surface of the films. Gelatin film alone without nanofiber reinforcement is difficult to handle due to the weakness of the thin membrane. Gelatin films with a fiber density as low as 3% v/v were structurally robust enough for handling, and manipulation into complex shapes. Mechanical testing confirmed that the addition of nanofibers enhanced the strength of gelatin films, in both dry and hydrated state. In vitro testing confirmed that nanofiber reinforced films were biocompatible and provided cells with directional guidance. Results demonstrate the promise of gelatin/PCL nanofiber composites as a tissue scaffolding material.     

In vitro investigations of a novel wound dressing concept based on biodegradable polyurethane

Abstract

Non-healing and partially healing wounds are an important problem not only for the patient but also for the public health care system. Current treatment solutions are far from optimal regarding the chosen material properties as well as price and source. Biodegradable polyurethane (PUR) scaffolds have shown great promise for in vivo tissue engineering approaches, but accomplishment of the goal of scaffold degradation and new tissue formation developing in parallel has not been observed so far in skin wound repair. In this study, the mechanical properties and degradation behavior as well as the biocompatibility of a low-cost synthetic, pathogen-free, biocompatible and biodegradable extracellular matrix mimicking a PUR scaffold was evaluated in vitro. The novel PUR scaffolds were found to meet all the requirements for optimal scaffolds and wound dressings. These three-dimensional scaffolds are soft, highly porous, and form-stable and can be easily cut into any shape desired. All the material formulations investigated were found to be nontoxic. One formulation was able to be defined that supported both good fibroblast cell attachment and cell proliferation to colonize the scaffold. Tunable biodegradation velocity of the materials could be observed, and the results additionally indicated that calcium plays a crucial role in PUR degradation. Our results suggest that the PUR materials evaluated in this study are promising candidates for next-generation wound treatment systems and support the concept of using foam scaffolds for improved in vivo tissue engineering and regeneration.     

A neurotrophic peptide-functionalized self-assembling peptide nanofiber hydrogel enhances rat sciatic nerve regeneration

Nerve guidance conduit (NGC) is a potential alternative to autologous nerve for peripheral nerve regeneration. A promising therapeutic strategy is to modify the nerve guidance conduit intraluminal microenvironment using physical and/or chemical guidance cues. In this study, a neurotrophic peptide-functionalized self-assembling peptide nanofiber hydrogel that could promote PC12 cell adhesion, proliferation, and neuronal differentiation in vitro was prefilled in the lumen of a hollow chitosan tube (hCST) to accelerate axonal regeneration in a rat sciatic nerve defect model. The functionalized self-assembling peptide was developed by introducing a neurotrophic peptide (RGI, RGIDKRHWNSQ) derived from brain-derived neurotrophic factor (BDNF) to the C-terminus of the self-assembling peptide RADA16-I (Ac-(RADA)₄-CONH₂). Morphological, histological, electrophysiological, and functional analyses demonstrated that the RGI-functionalized, self-assembling, peptide nanofiber hydrogel RAD/RGI could produce a neurotrophic microenvironment that markedly improved axonal regeneration with enhanced re-myelination and motor functional recovery.

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In vitro evaluation of electrospun gelatin–glutaraldehyde nanofibers

The gelatin–glutaraldehyde (gelatin–GA) nanofibers were electrospun in order to overcome the defects of ex-situ crosslinking process such as complex process, destruction of fiber morphology and decrease of porosity. The morphological structure, porosity, thermal property, moisture absorption and moisture retention performance, hydrolytic resistance, mechanical property and biocompatibility of nanofiber scaffolds were tested and characterized. The gelatin–GA nanofiber has nice uniform diameter and more than 80% porosity. The hydrolytic resistance and mechanical property of the gelatin–GA nanofiber scaffolds are greatly improved compared with that of gelatin nanofibers. The contact angle, moisture absorption, hydrolysis resistance, thermal resistance and mechanical property of gelatin–GA nanofiber scaffolds could be adjustable by varying the gelatin solution concentration and GA content. The gelatin–GA nanofibers had excellent properties, which are expected to be an ideal scaffold for biomedical and tissue engineering applications.     

Intervertebral disc (IVD): Structure, degeneration, repair and regeneration

Low back pain affects a large portion of the population, resulting in high care costs for therapy and treatment. One primary cause of low back pain is the degeneration of the intervertebral disc (IVD) resulting in the compression of the spinal nerves and adjacent vertebrae. Exact causes of degeneration are unknown, but it is thought that natural aging, and both biological and genetic factors may play a significant role in the degenerative process. Conventional methods to alleviate low back pain include spinal fusion and artificial disc replacement. Traditional treatments through spinal fusion may eliminate pain yet do not restore disc function and lead to further degeneration of adjacent levels by altering disc biomechanics and natural kinematics. Recently, artificial IVD replacements have started to gain interest, with two IVD implants currently approved in the United States. Although these implants facilitate the preservation of motions and disc space height, they are unable to sustain compressive forces due to their lack of elasticity. In addition, the implants may produce wear debris that can cause osteolysis and other deleterious effects. As an alternative to these conventional approaches, tissue engineered IVD constructs offer the advantage of biointegration while preserving the essential attributes of natural motion and disc space restoration. There is a great need for the development of tissue engineered scaffolds that simulate the natural 3D morphology and microenvironment of the targeted tissue. Scaffolds should facilitate biological transport to satisfy nutrition and waste removal requirements within the IVD. The discrete tissue architectures of the nucleus pulposus (NP) and annulus fibrosus (AF) have posed great challenges to IVD tissue engineering. Current attempts have not been able to satisfy the biological functions and/or mechanical properties of native tissue. Therefore, these current scaffolds are far from satisfactory. This review highlights the challenges met when tissue engineering an IVD, while offering a perspective on which biomaterials and scaffold fabrication methods have the realistic ability to serve as scaffolding constructs for IVD regeneration.