Intervertebral Disc Tissue Engineering Research Articles

Dose-dependent response of tissue-engineered intervertebral discs to dynamic unconfined compressive loading.

Because of the limitations of current surgical methods in the treatment of degenerative disc disease, tissue-engineered intervertebral discs (TE-IVDs) have become an important target. This study investigated the biochemical and mechanical responses of composite TE-IVDs to dynamic unconfined compression. TE-IVDs were manufactured by floating an injection molded alginate nucleus pulposus (NP) in a type I collagen annulus fibrosus (AF) that was allowed to contract for 2 weeks before loading. The discs were mechanically stimulated at a range of strain amplitude (1-10%) for 2 weeks with a duty cycle of 1 h on-1 h off-1 h on before being evaluated for their biochemical and mechanical properties. Mechanical loading increased all properties in a dose-dependent manner. Glycosaminoglycans (GAGs) increased between 2.8 and 2.2 fold in the AF and NP regions, respectively, whereas the hydroxyproline content increased between 1.2 and 1.8 fold. The discs also experienced a 2-fold increase in the equilibrium modulus and a 4.3-fold increase in the instantaneous modulus. Full effects for all properties were seen by 5% strain amplitude. These data suggest that dynamic loading increases the functionality of our TE-IVDs with region-dependent responses using a method that may be scaled up to larger disc models to expedite maturation for implantation.

In vitro characterization and in vivo behavior of human nucleus pulposus and annulus fibrosus cells in clinical-grade fibrin and collagen-enriched fibrin gels.

The intervertebral disc (IVD) presents a limited self-repair ability and cell-based therapies have been suggested to prevent or treat IVD lesions. Fibrin-based scaffolds as cell carriers are promising candidates in IVD tissue engineering, thanks to their ability to be easily delivered into the defect and to adapt to the lesion shape, to support/retain the injected cells into the implantation site and to favor the production of a suitable extracellular matrix (ECM). We evaluated the in vitro and in vivo behavior of human nucleus pulposus (NP) and annulus fibrosus (AF) cells in a clinical-grade collagen-enriched fibrin that has never been tested before for orthopedic applications, comparing it with clinical-grade fibrin. The survival of IVD cells seeded within fibrin or collagen-enriched fibrin and the ECM synthesis were evaluated by biochemical, immunohistochemical, and transcriptional analyses, prior and after subcutaneous implantation of the gels in nude mice. After 28 days of implantation, NP and AF cells were still detectable within explants, produced tissue-specific ECM, and showed a higher content of glycosaminoglycans (GAGs) and type I and II collagen compared to gels before implantation. Both the fibrin gels, enriched or not with collagen, seemed to be suitable for the culture of AF cells, being able to support the homogeneous synthesis of type I collagen, characteristic of the native fibrocartilaginous AF tissue. Differently, fibrin alone was a more suitable matrix for NP culture, supporting the homogeneous deposition of GAGs and type II collagen. In conclusion, our results suggest to combine AF cells with fibrin, enriched or not with collagen, and NP cells with fibrin alone to maintain the typical features of these cell populations, indicating these clinical-grade materials as viable options in cell-based treatments for IVD lesions.

Fibrin in intervertebral disc tissue engineering.

Fibrin is clinically employed as a versatile, safe, and clinically applicable sealant and cell carrier. It has been able to support disc cell survival, favor extracellular matrix production, and enhance the efficiency of cell transfer in the intervertebral disc (IVD). The aim of this review was to evaluate how fibrin has been used in vitro, in vivo, and in clinical trials for IVD tissue engineering. Within the in vitro studies, disc cells were cultured in fibrin alone or combined with other materials and a difference in the behavior of nucleus pulposus (NP) and annulus fibrosus (AF) cells was sometimes reported, but in general, the formation of fibrocartilaginous matrix was observed. Moreover, data concerning the fibrin long-term stability and its anti-inflammatory properties were found. Disc cells of human origin were never employed in combination with fibrin in vivo or in clinical trials. In vivo, disc degeneration models used to test the fibrin properties essentially involved NP injuries. The addition of cells, in particular if terminally differentiated, to the injected fibrin seemed to promote a more physiological matrix in comparison with fibrin alone. Important aspects should be further investigated in future studies such as the use of fibrin to treat AF lesions as well as the mechanical properties of the fibrin-based biomaterials and of the neoformed tissue. Finally, in vivo studies and clinical trials with in situ injection of fibrin and human disc cells should be performed.

Tissue-Engineered Intervertebral Discs: In Vivo Outcome in the Rodent Spine

Tissue-Engineered Intervertebral Discs: In Vivo Outcome in the Rodent Spine

Hif-αs Modulation of Sox9-Dependent Extracellular Matrix Production: Insight into Intervertebral Disc Cell Activity under Hypoxia

Introduction Intervertebral disc (IVD) degeneration leads to loss of mechanical function of motion segment and is attributed to deregulated extracellular matrix components in the nucleus pulposus (NP). The NP is rich in type II collagen (encoded by COL2A1) and aggrecan (encoded by AGC1), which are known to be regulated by the master chondrogenic transcription factor, Sox9.1 In situ studies of human degenerated NP illustrated a reduction of COL2A1 and absence of AGC1 expression, whereas SOX9 expression level remained high.2 This indicates that there may be other factors in the IVD that can regulate the Sox9-dependent matrix expression. Recent studies in aged rabbit IVD suggest that the abnormal NP cells may be contributed by a migration of the Sox9-expressing prechondrocytic cells from the annulus fibrosus (AF) adjacent to the endplate (EP).3, 4 Among the hypoxia inducible factor-α subunits (Hif-α), Hif-1α and Hif-2α (EPAS1) have been illustrated as important transcription factors in maintaining disc cell and matrix homeostasis, particularly in the hypoxic NP region.5-7 We hypothesized that Hif-αs may regulate the Sox9-dependent transcription of the extracellular matrix genes in response to IVD degeneration. First, we aimed at identification of Sox9 and Hif-αs co-expression in cells of non-degenerated and degenerated IVD. Second, we tested the modulatory effects of Hif-1α/Hif-2α on the expression of Col2a1 and Agc1 using Sox9-expressing mouse prechondrocytic cells as a model. Materials and Methods IVD were harvested from wild-type C57BL/6N mice at 3 and 6 month old. Disc degeneration was induced by an insertion of 25G needle into contralateral AF of 4-month old Lewis rat and discs were harvested post 2 to 4 weeks of operation. Lumbar IVD of scoliosis patients and degenerative disc disease patient were collected. All animal and human works were approved by local ethical committee. Comparative immunohistochemistry staining of Hif-1α, Hif-2α and Sox9 were studied in parrafilm sections of IVD. Luciferase-based promoter assays based on specific Col2a1 or Agc1 cis-acting elements via the overexpression for each of the HIF-α subunits and/or for Sox9 in ATDC5 cells were performed. Results Relative co-expression patterns of Hif-1α, Hif-2α and Sox9 were detected in AF, NP and EP of the mouse, rat and human IVD. Intracellular expression of Hif-1α, Hif-2α, and Sox9 were confirmed by immunofluorescence, indicating a possible role for Hif-αs-Sox9 tonic activity in the IVD. By luciferase-based promoter assay, Sox9 is demonstrated as a dominant transcription factor in the activation of transcription of Col2a1 and Agc1 in mouse chondroprogenitor cells. Overt additive transcriptional upregulation of Col2a1 and Agc1 was observed with a co-expression of HIF-1α and Sox9. Strikingly, Hif-2α inhibited the Sox9-dependent transcriptional upregulation of Col2a1 and Agc1. Conclusion Our findings indicate the presence of Hif-αs/Sox9 expressing cells in IVD. In addition, our data suggest that Hif-αs can modulate Sox9-mediated transcriptional regulation of Col2a1 and Agc1 in prechondrocytic cells, implicating a possible role of dynamic Hif-αs-Sox9 interaction in the IVD degeneration process. Further investigations of the orchestration mechanisms of Hif-αs/Sox9 in the IVD may provide insights in potential strategies to alleviate disc degeneration. Disclosure of Interest None declared References Richardson SM, Hoyland JA, Mobasheri R, Csaki C, Shakibaei M, Mobasheri A. Mesenchymal stem cells in regenerative medicine: opportunities and challenges for articular cartilage and intervertebral disc tissue engineering. J Cell Physiol 2010;222(1):23–32 Sive JI, Baird P, Jeziorsk M, Watkins A, Hoyland JA, Freemont AJ. Expression of chondrocyte markers by cells of normal and degenerate intervertebral discs. Mol Pathol 2002;55(2):91–97 Henriksson H, Thornemo M, Karlsson C, et al. Identification of cell proliferation zones, progenitor cells and a potential stem cell niche in the intervertebral disc region: a study in four species. Spine 2009;34(21):2278–2287 Henriksson HB, Svala E, Skioldebrand E, Lindahl A, Brisby H. Support of concept that migrating progenitor cells from stem cell niches contribute to normal regeneration of the adult mammal intervertebral disc: a descriptive study in the New Zealand white rabbit. Spine 2012;37(9):722–732 Rajpurohit R, Risbud MV, Ducheyne P, Vresilovic EJ, Shapiro IM. Phenotypic characteristics of the nucleus pulposus: expression of hypoxia inducing factor-1, glucose transporter-1 and MMP-2. Cell Tissue Res 2002;308(3):401–407 Risbud MV, Guttapalli A, Stokes DG, et al. Nucleus pulposus cells express HIF-1 alpha under normoxic culture conditions: a metabolic adaptation to the intervertebral disc microenvironment. J Cell Biochem 2006;98(1):152–159 Agrawal A, Gajghate S, Smith H, et al. Cited2 modulates hypoxia-inducible factor-dependent expression of vascular endothelial growth factor in nucleus pulposus cells of the rat intervertebral disc. Arthritis Rheum 2008;58(12):3798–3808

Tissue-engineered intervertebral discs: MRI results and histology in the rodent spine

Object Tissue-engineered intervertebral discs (TE-IVDs) represent a new experimental approach for the treatment of degenerative disc disease. Compared with mechanical implants, TE-IVDs may better mimic the properties of native discs. The authors conducted a study to evaluate the outcome of TE-IVDs implanted into the rat-tail spine using radiological parameters and histology. Methods Tissue-engineered intervertebral discs consist of a distinct nucleus pulposus (NP) and anulus fibrosus (AF) that are engineered in vitro from sheep IVD chondrocytes. In 10 athymic rats a discectomy in the caudal spine was performed. The discs were replaced with TE-IVDs. Animals were kept alive for 8 months and were killed for histological evaluation. At 1, 5, and 8 months, MR images were obtained; T1-weighted sequences were used for disc height measurements, and T2-weighted sequences were used for morphological analysis. Quantitative T2 relaxation time analysis was used to assess the water content and T1ρ-relaxation time to assess the proteoglycan content of TE-IVDs. Results Disc height of the transplanted segments remained constant between 68% and 74% of healthy discs. Examination of TE-IVDs on MR images revealed morphology similar to that of native discs. T2-relaxation time did not differ between implanted and healthy discs, indicating similar water content of the NP tissue. The size of the NP decreased in TE-IVDs. Proteoglycan content in the NP was lower than it was in control discs. Ossification of the implanted segment was not observed. Histological examination revealed an AF consisting of an organized parallel-aligned fiber structure. The NP matrix appeared amorphous and contained cells that resembled chondrocytes. Conclusions The TE-IVDs remained viable over 8 months in vivo and maintained a structure similar to that of native discs. Tissue-engineered intervertebral discs should be explored further as an option for the potential treatment of degenerative disc disease.

Establishment of a Promising Human Nucleus Pulposus Cell Line for Intervertebral Disc Tissue Engineering

Low-back pain caused by intervertebral disc degeneration could be recovered by the regeneration of the nucleus pulposus (NP). This study aimed to establish a chondrogenic recovery model with promising a human NP (hNP) cell line, an immortalized hNP (ihNP), which could be a screening platform to identify regenerative drugs. The ihNP cells were created from primary human NP cells transfected with a retroviral vector-driven HPV16 E6/E7. Growth properties and characteristics of ihNP were evaluated by comparing with parental NP cells. Successful immortalization of ihNP cells stably expressed HPV 16 E6/E7 mRNA. The doubling time of ihNP was shortened to 53.16±2.63 h compared with parental hNP-P1. Cell cycle regulators, including p53, p21, and pRB were downregulated compared to parental hNP-P1. The in vivo neoplastic forming assay also demonstrated that the ihNP was nontumorigenic. After 25 generations of cell cultures, the ihNP cells, yet stably expressed chondrogenic genes, including (SOX9), type II collagen (Col II), aggrecan, decorin, biglycan, and versican. Higher expressions of chondrogenic proteins, including Col II, phosphorylated SOX9 (p-SOX9), and CD44 were also determined. Under the stressful inflammatory conditions induced by lipopolysaccharides (LPS), the regenerative and anti-inflammatory potentials of ihNP in two-dimensional culture with the presence of platelet-rich plasma (PRP) were evaluated by reverse transcriptase polymerase chain reaction. PRP showed significant effects on restoring diminished chondrogenic markers and deleterious inflammatory responses induced by LPS in ihNP. The therapeutic potentials of ihNP in three-dimensional neocartilage model could also be exerted by PRP using histological evaluation and immunological staining. Hence, the established ihNP cells can provide a chondrogenic recovery model as a regenerative drug screening tool for further regenerative drug discovery and development.

A novel integrated biphasic silk fibroin scaffold for intervertebral disc tissue engineering

The intervertebral disc (IVD) tissue engineering construct provides a promising approach for the treatment of symptomatic degenerative disc diseases. In this study, a novel 3D silk fibroin biphasic scaffold for integrated IVD tissue engineering was fabricated by sequentially using the paraffin sphere-leaching method for annulus fibrosus (AF) phase and the phase separation method for nucleus pulposus (NP) phase. Both phases were perfectly integrated and possessed a highly interconnected porous structure with pore size of 220±23.1μm for AF and 90±17.8μm for NP phase. Furthermore, the scaffolds were found to have a high porosity of 91% and 93% for AF and NP phases, respectively. In addition, this silk biphasic scaffold had a relative high compressive modulus under wet conditions (150.7±6.8kPa). Rabbit AF and NP cells could attach, grow and further penetrate into the scaffold and distribute uniformly. This silk fibroin biphasic IVD scaffold emerges as a potential candidate for IVD tissue engineering.

Stem Cell Regeneration of Degenerated Intervertebral Discs: Current Status (Update)

Low back pain, strongly associated with intervertebral disc (IVD) degeneration, affects a large proportion of the population and has major social and economic costs. Current treatments remain inadequate, targeting the symptoms without addressing the underlying cause. As such, efforts are being directed towards development of therapies aimed at alleviating pain through the restoration of IVD function. The potential of cell-based therapies for the treatment of IVD degeneration are being actively explored, with an emphasis on cell/biomaterial tissue engineering. Adult mesenchymal stem cells, capable of differentiating down the discogenic lineage, have shown promise as a suitable cell source for IVD tissue engineering. However, a number of factors, (discussed in this review), remain to be addressed, including development of a differentiation protocol to produce the correct cell phenotype, identification of suitable biomaterials for cell delivery/implantation, and ensuring cell survival and correct function upon implantation into the degenerate IVD.

Tissue-Engineered Intervertebral Discs: In Vivo Outcome in the Rodent Spine

IntroductionIntervertebral (IVD) disc degeneration is characterized by a cascade of structural, chemical, and cellular changes. The current standard for surgical treatment of degenerated discs invo...

A novel natural ECM-derived biphasic scaffold for intervertebral disc tissue engineering

The tissue-engineered intervertebral disc (IVD) construct offers an alternative therapeutic possibility for symptomatic degenerative disc diseases. A porcine-derived acellular matrix as an integrated biphasic IVD scaffold was fabricated using an optimal decellularization procedure with a simple freeze-drying and cross-linking technique. The biphasic construct contained annulus fibrosus (AF) phase with cancellous bone matrix gelatin (CBMG) and the nucleus pulposus (NP) phase with cartilage extracellular matrix (CECM). In addition, this decellularized IVD scaffold possessed interconnected and tightly integrated porous microstructure (pore size of AF and NP phase at 401.4±13.1μm and 231.6±57.2μm, respectively), and had appropriate mechanical property. Histological results showed no residual cells remaining within the decellularized scaffold but abundant collagen proteins retained. Fluorescence microscope and scanning electron microscopy demonstrated that the novel scaffold could provide a suitable 3-D environment to support adhesion and proliferation of both AF and NP cells. In conclusion, this xenogenous ECM-derived IVD scaffold provides potential as an alternative for IVD tissue engineering.

Normal and degenerated rabbit nucleus pulposus cells in in vitro cultures: A biological comparison

This study examined the biological characteristics of normal and degenerated rabbit nucleus pulposus (NP) cells in vitro in order to provide seed cells for intervertebral disc (IVD) tissue engineering. A total of 8 adult New Zealand white rabbits underwent annulus puncture to establish models of intervertebral disc degeneration (IDD). Four weeks later, normal and degenerated NP cells were obtained. Cell morphology was observed by light and electron microscopy. Cell viability was measured by MTT assay. Cell cycle and expression of extracellular matrix (ECM)-related genes (aggrecan and type II collagen) were determined by using flow cytometry and RT-PCR respectively. The growth curve of normal NP cells showed that the cells at passage 4 tended to slowly grow on the fifth day of culture. The density of normal NP cells at passages 5 to 7 was significantly less than that of the first-passage cells 2 or 3 days after seeding (P<0.05). The degenerated NP cells at passage 3 showed slow growth at 4th day. After 5 passages, the degenerated NP cells assumed stagnant growth and the growth seemed to stop at passage 7. The MTT assay revealed that for both normal and degenerated NP cells, the absorbance (A) value at passages 4-7 was obviously decreased as compared with that at passage 1 (P<0.05). Cell cycle analysis showed that the proportion of normal NP cells at Gl phase was 65.4%±3.5%, significantly lower than that of degenerated NP cells at the same cell cycle phase with the value being 77.6%±4.8%. The degenerated NP cells were predominantly arrested at G1 phase and failed to enter S phase. The expression of type II collagen and aggrecan was significantly decreased with passaging. It was concluded that normal NP cells possessed good viability and proliferative capacity by the third passage, and they could secrete large amounts of ECM within this period. The normal NP cells may serve as seed cells for IVD tissue engineering.

Fabrication and Evaluation of a Novel Integrated Annulus Fibrosus-Nucleus Pulposus Hybrid Scaffold

The scope of this study was to fabricate and evaluate a novel integrated annulus fibrosus-nucleus pulposus scaffold composed of bone matrix gelatin (BMG) and cartilage extracellular matrix (ECM) respectively. Scaffolds were fabricated by a serial physicochemical and free-drying process. The physiochemical property and compatibility of the composite scaffold with intervertebral disc cells were evaluated. HE staining showed no residual cells in both annulus fibrosus and nucleus pulposus scaffolds. SEM observation revealed that the integration of annulus fibrosus region and nucleus pulposus region was well. The pore diameter of annulus fibrosus scaffold is about 401.4±13.1 μm and the nucleus pulposus scaffold 112.4±21.8 μm, which demonstrated that the composite scaffolds possess interconnected pores structure. The average compressive elastic modulus of the integrated scaffold was 49.06 ±15.57 kpa, which was lower than that of porcine coccyx intervertebral disc, 135.9±28.9 kPa. SEM observation and LIVE/DEAD staining showed good adhesion and high viability of intervertebral disc cells in the integrated scaffold. In conclusion, the integrated annulus fibrosus-nucleus pulposus scaffold can be used for intervertebral disc tissue engineering because of its non-immunogenicity, preservation of ECM composition, good physicochemical property and high biocompatibility with interbertebral disc cells.

Effect of a synthetic link N peptide nanofiber scaffold on the matrix deposition of aggrecan and type II collagen in rabbit notochordal cells

Self-assembling peptide nanofiber scaffolds have been studied extensively as biological materials for 3-dimensional cell culture and repairing tissue defects in animals. However, few studies have applied peptide nanofiber scaffolds in the tissue engineering of intervertebral discs (IVDs). In this study, a novel functionalized peptide scaffold was specifically designed for IVD tissue engineering, and notochordal cells (NCs) as an alternative cell source for IVD degeneration were selected to investigate the bioactive scaffold material. The novel RADA16-Link N self-assembling peptide scaffold material was designed by direct coupling to a bioactive motif link N. The link N nanofiber scaffold (LN-NS) material was obtained by mixing pure RADA16-I and RADA16-Link N (1:1) designer peptide solutions. Although live/dead cell assays showed that LN-NS and RADA16-I scaffold materials were both biocompatible with NCs, the LN-NS material significantly promoted NC adhesion compared with that of the pure RADA16-I SAP scaffold material. The depositions of aggrecan and type II collagen, which are significant markers for IVD cells, were remarkably increased. Furthermore, the results indicated that the link N motif, the matrix analog of the nucleus pulposus, significantly promoted the accumulation of other extracellular matrices in vitro. We conclude that the novel LN-NS material is a promising biological scaffold material, and may have a broad range of applications in IVD tissue engineering.

Chemical modification of collagen improves glycosaminoglycan retention of their co-precipitates

Being prevalent extracellular matrix components, collagen and glycosaminoglycan (GAG) are co-precipitated as scaffolds for tissue regeneration. However, the amount of GAG incorporated and its long-term retention present a persistent problem. In this study, chemical modifications, namely deamination, methylation and amination, were used to alter the net charge of collagen prior to fabrication of collagen–GAG co-precipitate. While most GAGs were lost in the untreated group and the deaminated group within 1day, methylation and amination of collagen retained over 20% and 40% GAG after 6days, respectively. Moreover, over 60% of GAG retention was achieved in the aminated group after cell seeding for 8days. Furthermore, amination of collagen increased the GAG/hydroxyproline ratio in the co-precipitate to >4.5, approaching that of native nucleus pulposus. Ultrastructural analysis showed that the aminated group contains abundant granular substances resembling the extracellular matrix of native nucleus pulposus. Despite lower initial cell adhesion than untreated, all modified scaffolds promoted proliferation of human mesenchymal stem cells (hMSCs) and showed >95% cell viability at all time points. Cell morphology was distinct among the different groups, being round in the untreated control and methylated groups but elongated in deaminated and aminated groups. hMSCs adhered to scaffolds via collagen receptor integrin α2β1 in all groups, while all but the aminated group showed extensive expression of the general matrix receptor integrin αv. This work reports an effective method, namely amination of collagen, to improve GAG incorporation and retention in collagen–GAG co-precipitates, facilitating the fabrication of GAG-rich collagenous scaffold for intervertebral disc tissue engineering.

Combination of MRI and 99mTc-NTP 15-5 Scintigraphy for the Assessment of Intervertebral Disk Degeneration and Tissue Engineering

Combination of MRI and 99mTc-NTP 15-5 Scintigraphy for the Assessment of Intervertebral Disk Degeneration and Tissue Engineering

Combination of MRI and 99mTc-NTP 15-5 Scintigraphy for the Assessment of Intervertebral Disk Degeneration and Tissue Engineering

Introduction Intervertebral disk degeneration is one of the leading causes of low back pain. The development of regenerative cell-based biotherapies for degenerated IVD has recently been contemplated. To evaluate the preclinical relevance of such therapies, animal models combined with longitudinal imaging methods such as MRI are currently used. However, facing the limitations of MRI and to further decipher the early process of IVD degeneration, innovative radio imaging approaches are considered promising. In this context, we were interested in determining whether a scintigraphic imaging approach using a specific proteoglycan radiotracer (99mTc-NTP 15-5) could be instrumental to assess the biofunctionality of IVD extracellular matrix. Materials and Methods New Zealand white rabbits (1-, 6-, 12- and 30-month-old; n = 3 for each age) were used. Early aging of lumbar IVD was determined by MRI and scintigraphic analysis. MRI images were analyzed using T2-weighted signal intensity (T2wsi) and scored according to the Pfirrmann grading. In a first set of experiments, scintigraphic analyses were performed to assess the kinetic distribution in 12-month old rabbits. Ratio IVD on muscle fixation was measured. The distribution of 99 mTc-NTP-15-5 in IVD was evaluated by in vivo scintigraphic imaging (BigSpace Gamma Imager) on three rabbits of each age. Results MRI assessments of rabbit IVDs reveal as a function of age, a grading decrease in IVD signal intensity (T2wsi) associated with an increase in Pfirrmann grade. These data indicate the existence of an aging/degeneration process of rabbit IVD. As a function of escalating ages, a differential uptake of 99 mTc-NTP 15-5 in IVD was observed. Uptake was maximal at 1 month of age, and was followed by a dramatic decrease as early as 6 months (approximately 50% of radioactive signal). After 6 months, the intensity of decrease was less pronounced (approximately 10% of radioactive signal). Conclusion These in vivo results highlight the interest of combining 99 mTc-NTP-15-5 scintigraphic imaging of proteoglycans with MRI. Whereas MRI T2wsi reflects the hydration level, 99 mTc-NTP-15-5 scintigraphy allowed the functional assessment of IVD at the proteoglycan level. Such multi-imaging approach could be useful for the longitudinal study of engineered IVD following the intradiscal transplantation of mesenchymal stem cells using an injectable biomaterial. In addition, the use of scintigraphy imaging in human should be paid further attention particularly to detect the early IVD degeneration. I confirm having declared any potential conflict of interest for all authors listed on this abstract Yes Disclosure of Interest None declared

Collagen-Glycosaminoglycan Coprecipitate and Photochemically Cross-Linked Collagen for Disk Replacement

IntroductionTraditional treatments of severe disk degeneration such as discectomy were not satisfactory. Recent research focuses on developing biological substitutes, using tissue engineering methods, for nucleus pulposus and annulus fibrosus.1,2 A few studies had demonstrated the fabrication of biphasic composite scaffold as nucleus and annulus replacements using alginate and polyglycolic acid and alginate and collagen.3,4 Being the prevalent matrix components in native disc, collagen and glycosaminoglycans (GAGs) are potentially suitable biomaterials for a more nature-simulating scaffold. Previously, we have demonstrated that chemical modifications of collagen by deamination, methylation, and amination were able to control the GAG binding to and retention from the collagen meshwork, resulting in controllable GAG:HYP ratio, while maintaining over 90% cell viability. In this study, the ultrastructure of the coprecipitates fabricated in acidic and neutral pH was evaluated. Moreover, aminated CG coprecipitate, with optimal GAG:HYP ratio, along with mechanically strengthened photochemically crosslinked collagen lamellae previously developed in our lab, were used to fabricate a biphasic composite scaffold. On-going work is underway to test the cytocompatibility and mechanical properties of the composite scaffold. Materials and MethodsFirst, modified collagens (deaminated, aminated, and methylated) were coprecipitated with GAG to form CG coprecipitates, which were observed under SEM before and after neutralization. Second, aminated CG was entrapped as the core of a collagen shell, which was photochemically crosslinked in the presence of rose bengal and argon laser before partial dehydration of the whole biphasic scaffold. Rabbit mesenchymal stem cells would be cultured in the core of the scaffold for 7 days. Scaffolds would be evaluated by immunohistology and cell survival was visualized with live/dead staining. The scaffold would be tested for creep and dynamic responses. ResultsFirst, collagen fibers in modified CGs had distinctive ultrastructural features at different pH. In acidic conditions, fibers were compact with small meshes (∼50 nm) in aminated, methylated, and nontreated CGs. In physiological pH, observations in the three CGs were also distinctive. Meshsize increased in non-treated CG. Sheet-like substance wrapped around the fibers in methylated CG. Granule-like ground substances were found in aminated CG. Seoncd, the feasibility of fabricating a biphasic composite scaffold with aminated CG as the core and the photochemically cross-linked collagen lamellae as the shell has been demonstrated. Preliminary mechanical study demonstrated that a step strain of 72% (pre-strain included) resulted in resistance stress up to 1 MPa, which gradually decreased to 0.5 MPa over 20 minutes. ConclusionThis study demonstrated that there were structural differences between different modified CGs and the fabrication of a biphasic collagen-GAG-based composite scaffold for IVD tissue engineering is feasible. The functional features of the composite scaffold have to be further evaluated.I confirm having declared any potential conflict of interest for all authors listed on this abstractYesDisclosure of InterestNone declaredSebastineM, Williams DJ. Current developments in tissue engineering of nucleus pulposus for the treatment of intervertebral disk degeneration,” Conference proceedings?: Annual International Conference of the IEEE Engineering in Medicine and Biology Society.IEEE Engineering in Medicine and Biology Society. Conference 2007;2007:6401–6406Nerurkar NL, Baker BM, Sen S, Wible EE, Elliott DM, Mauck RL. Nanofibrous biologic laminates replicate the form and function of the annulus fibrosus. Nature materials 2009;8(12):986–992Mizuno H, Roy AK, Zaporojan V, Vacanti CA, Ueda M, Bonassar LJ. Biomechanical and biochemical characterization of composite tissue-engineered intervertebral discs. Biomaterials 2006;27(3):362–370Bowles RD, Williams RM, Zipfel WR, Bonassar LJ. Self-assembly of aligned tissue-engineered annulus fibrosus and intervertebral disk composite via collagen gel contraction. Tissue engineering. Part A 2010;16(4):1339–1348

Collagen-Glycosaminoglycan Coprecipitate and Photochemically Cross-Linked Collagen for Disk Replacement

Introduction Traditional treatments of severe disk degeneration such as discectomy were not satisfactory. Recent research focuses on developing biological substitutes, using tissue engineering methods, for nucleus pulposus and annulus fibrosus.1,2 A few studies had demonstrated the fabrication of biphasic composite scaffold as nucleus and annulus replacements using alginate and polyglycolic acid and alginate and collagen.3,4 Being the prevalent matrix components in native disc, collagen and glycosaminoglycans (GAGs) are potentially suitable biomaterials for a more nature-simulating scaffold. Previously, we have demonstrated that chemical modifications of collagen by deamination, methylation, and amination were able to control the GAG binding to and retention from the collagen meshwork, resulting in controllable GAG:HYP ratio, while maintaining over 90% cell viability. In this study, the ultrastructure of the coprecipitates fabricated in acidic and neutral pH was evaluated. Moreover, aminated CG coprecipitate, with optimal GAG:HYP ratio, along with mechanically strengthened photochemically crosslinked collagen lamellae previously developed in our lab, were used to fabricate a biphasic composite scaffold. On-going work is underway to test the cytocompatibility and mechanical properties of the composite scaffold. Materials and Methods First, modified collagens (deaminated, aminated, and methylated) were coprecipitated with GAG to form CG coprecipitates, which were observed under SEM before and after neutralization. Second, aminated CG was entrapped as the core of a collagen shell, which was photochemically crosslinked in the presence of rose bengal and argon laser before partial dehydration of the whole biphasic scaffold. Rabbit mesenchymal stem cells would be cultured in the core of the scaffold for 7 days. Scaffolds would be evaluated by immunohistology and cell survival was visualized with live/dead staining. The scaffold would be tested for creep and dynamic responses. Results First, collagen fibers in modified CGs had distinctive ultrastructural features at different pH. In acidic conditions, fibers were compact with small meshes (∼50 nm) in aminated, methylated, and nontreated CGs. In physiological pH, observations in the three CGs were also distinctive. Meshsize increased in non-treated CG. Sheet-like substance wrapped around the fibers in methylated CG. Granule-like ground substances were found in aminated CG. Seoncd, the feasibility of fabricating a biphasic composite scaffold with aminated CG as the core and the photochemically cross-linked collagen lamellae as the shell has been demonstrated. Preliminary mechanical study demonstrated that a step strain of 72% (pre-strain included) resulted in resistance stress up to 1 MPa, which gradually decreased to 0.5 MPa over 20 minutes. Conclusion This study demonstrated that there were structural differences between different modified CGs and the fabrication of a biphasic collagen-GAG-based composite scaffold for IVD tissue engineering is feasible. The functional features of the composite scaffold have to be further evaluated. I confirm having declared any potential conflict of interest for all authors listed on this abstract Yes Disclosure of Interest None declared SebastineM, Williams DJ. Current developments in tissue engineering of nucleus pulposus for the treatment of intervertebral disk degeneration,” Conference proceedings?: Annual International Conference of the IEEE Engineering in Medicine and Biology Society.IEEE Engineering in Medicine and Biology Society. Conference 2007;2007:6401–6406 Nerurkar NL, Baker BM, Sen S, Wible EE, Elliott DM, Mauck RL. Nanofibrous biologic laminates replicate the form and function of the annulus fibrosus. Nature materials 2009;8(12):986–992 Mizuno H, Roy AK, Zaporojan V, Vacanti CA, Ueda M, Bonassar LJ. Biomechanical and biochemical characterization of composite tissue-engineered intervertebral discs. Biomaterials 2006;27(3):362–370 Bowles RD, Williams RM, Zipfel WR, Bonassar LJ. Self-assembly of aligned tissue-engineered annulus fibrosus and intervertebral disk composite via collagen gel contraction. Tissue engineering. Part A 2010;16(4):1339–1348

Demineralized Bone Particle Impregnated Poly(l-Lactide-co-Glycolide) Scaffold for Application in Tissue-Engineered Intervertebral Discs

Demineralized bone particle (DBP) contains powerful bioactive molecules that facilitate new bone or cartilage growth. We developed hybrid scaffolds of poly(l-lactide-co-glycolide) (PLGA) with various concentrations of DBP (DBP/PLGA), of which phenotypes on intervertebral disc (IVD) cells were investigated. The hybrid scaffold has a cylindrical donut shape with two distinct parts; the inner is for the nucleus pulposus (NP) and the outer is for annulus fibrosus (AF). Rabbit NP and AF cells were seeded into the inner and outer regions of the DBP/PLGA scaffolds separately. Disc cell viability in DBP/PLGA scaffolds was superior to pure PLGA scaffold and increased with increasing DBP concentration. In vitro- and in vivo-formed tissues were characterized by RT-PCR, Safranin-O, Masson’s trichrome staining and immunohistochemi- cal staining for type-I and type-II collagen. DBP/PLGA hybrid scaffolds revealed more active expression of disc phenotypes, as characterized by protein and mRNA expression, than the PLGA control. This study provides valuable information for potential disc replacement using DBP and PLGA.