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Engineered bone tissue synthesized from mesenchymal stem cell progenitors has numerous applications throughout the fields of regenerative medicine and tissue engineering. However, these multipotent cells offer little tissue-building assistance without differentiation direction from environmental cues such as bone morphogenetic proteins (BMPs). Unfortunately, BMP dosing and environmental cues can be difficult to control bothin vitroand afterin vivodelivery. Several BMP antagonists are expressed by cells in response to BMP dosing that bind extracellular BMPs and reduce their effective concentration. Here, we use CRISPR-guided gene-modulation technology to downregulate the expression of three BMP antagonists, noggin, gremlin-1, and gremlin-2, in adipose-derived stem cells (ASCs). We show that regulating noggin using this method results in ASC osteogenesis without the need for exogenous growth factors. To demonstrate the versatility and the precision capabilities of these engineered cells, we employ them with CRISPRa multiplex-engineered chondrogenic cells as a proof-of-concept tissue engineering application by creating a tissue gradient similar to the fibrocartilage-to-mineralized-fibrocartilage gradient in the tendon/ligament enthesis or intervertebral disc attachment. In doing so, we show that multiple CRISPR multiplex engineered cell types can be utilized in concert to provide a high degree of tissue developmental control without the use of exogenous growth factors.

Stem cell therapies have shown promise for regenerative treatment for musculoskeletal conditions, but their success is mixed. To enhance regenerative effects, growth factors are utilized to induce differentiation into native cell types, but uncontrollable in vivo conditions inhibit differentiation, and precise control of expressed matrix proteins is difficult to achieve. To address these issues, we investigated a novel method of enhancing regenerative phenotype through direct upregulation of major cartilaginous tissue proteins, aggrecan (ACAN), and collagen II (COL2A1) using dCas9-VPR CRISPR gene activation systems. We demonstrated increased expression and deposition of targeted proteins independent of exogenous growth factors in pellet culture. Singular upregulation of COL2A1/ACAN interestingly indicates that COL2A1 upregulation mediates the highest sulfated glycosaminoglycan (sGAG) deposition, in addition to collagen II deposition. Through RNA-seq analysis, this was shown to occur by COL2A1 upregulation mediating broader chondrogenic gene expression changes. Multiplex upregulation of COL2A1 and ACAN together resulted in the highest sGAG, and collagen II deposition, with levels comparable to those in chondrogenic growth factor-differentiated pellets. Overall, this work indicates dCas9-VPR systems can robustly upregulate COL2A1 and ACAN deposition without growth factors, to provide a novel, precise method of controlling stem cell phenotype for cartilage and intervertebral disc cell therapies and tissue engineering. IMPACT STATEMENT: Stem cell therapies have come about as a potential regenerative treatment for musculoskeletal disease, but clinically, they have mixed results. To improve stem cell therapies, growth factors are used to aid a regenerative cell phenotype, but their effects are inhibited by in vivo musculoskeletal disease environments. This article describes CRISPR gene activation-based cell engineering methods that provide a growth factor-free method of inducing chondrogenic extracellular matrix deposition. This method is demonstrated to be as/more potent as growth factors in inducing a chondrogenic phenotype in pellet culture, indicating potential utility as a method of enhancing stem cell therapies for musculoskeletal disease.

Low back pain is among the leading causes of disability worldwide. The degenerative intervertebral disc (IVD) environment contains pathologically high levels of inflammatory cytokines and acidic pH hypothesized to contribute to back pain by sensitizing nociceptive neurons to stimuli that would not be painful in healthy patients. We hypothesized that the degenerative IVD environment drives discogenic pain by sensitizing nociceptive neurons to mechanical loading. To test this hypothesis, we developed an in vitro model that facilitated the investigation of interactions between the degenerative IVD environment, nociceptive neurons innervating the IVD and mechanical loading of the disc; and, the identification of the underlying mechanism of degenerative IVD induced nociceptive neuron sensitization. In our model, rat dorsal root ganglia (DRG) neurons were seeding onto bovine annulus fibrosus tissue, exposed to degenerative IVD conditioned media and/or acidic pH, and subjected to cyclic tensile strain (1 Hz; 1%-6% strain) during measurement of DRG sensory neuron activity via calcium imaging. Using this model, we demonstrated that both degenerative IVD conditioned media and degenerative IVD acidic pH levels induced elevated nociceptive neuron activation in response to physiologic levels of mechanical strain. In addition, interleukin 6 (IL-6) was demonstrated to mediate degenerative IVD conditioned media induced elevated nociceptive neuron activation. These results demonstrate IL-6 mediates degenerative IVD induced neuron sensitization to mechanical loading and further establishes IL-6 as a potential therapeutic target for the treatment of discogenic pain. Data further suggests the degenerative IVD environment contains multiple neuron sensitization pathways (IL-6, pH) that may contribute to discogenic pain.

Back pain is the leading cause of disability worldwide and contributes to significant socioeconomic impacts. It has been hypothesized that the degenerative intervertebral disc (IVD) contributes to back pain by sensitizing nociceptive neurons innervating the IVD to stimuli that would not be painful to healthy patients. However, the inflammatory signaling networks mediating this sensitization remain poorly understood. A better understanding of the underlying mechanisms of degenerative IVD-induced changes in nociception is required to improve the understanding and treatment of back pain. Toward these ends, a novel in vitro model was developed to investigate degenerative IVD-induced changes in dorsal root ganglion (DRG) neuron activation by measuring DRG neuron activity following neuron seeding on human degenerative IVD tissue collected from patients undergoing surgical treatment for back pain. Lentiviral clustered regularly interspaced palindromic repeat (CRISPR) epigenome editing vectors were built to downregulate the inflammatory receptors TNFR1, IL1R1, and IL6st in DRG neurons in single- and multiplex. Multiplex CRISPR epigenome editing of inflammatory receptors demonstrated that degenerative IVD tissue drives thermal sensitization through the simultaneous and redundant signaling of interleukin (IL)-6, tumor necrosis factor alpha (TNF-α), and IL-1β. This work elucidates redundant signaling pathways in neuron interactions with the degenerative IVD and suggests the need for multiplex targeting of IL-6, TNF-α, and IL-1β for pain modulation in the degenerative IVD.

Cell therapies are an up and coming technology in orthopedic medicine that has the potential to provide regenerative treatments for musculoskeletal disease. Despite numerous cell therapies showing preclinical success for common musculoskeletal indications of disc degeneration and osteoarthritis, there have been mixed results when testing these therapies in humans during clinical trials. A theory behind the mixed success of these cell therapies is that the harsh microenvironments of the disc and knee they are entering inhibit their anabolism and survival. Therefore, there is much ongoing research looking into how to improve the survival and anabolism of cell therapies within these musculoskeletal disease environments. This includes research into improving cell function under specific microenvironmental conditions known to exist in the intervertebral disc (IVD) and knee environment such as hypoxia, low-nutrient conditions, hyperosmolarity, acidity, and inflammation. This research also includes improving differentiation of cells into desired native cell phenotypes to better enhance their survival and anabolism in the knee and IVD. This review highlights the effects of specific musculoskeletal microenvironmental challenges on cell therapies and what research is being done to overcome these challenges. Impact statement While there has been significant clinical interest in using cell therapies for musculoskeletal pathologies in the knee and intervertebral disc, cell therapy clinical trials have had mixed outcomes. The information presented in this review includes the environmental challenges (i.e., acidic pH, inflammation, hyperosmolarity, hypoxia, and low nutrition) that cell therapies experience in these pathological musculoskeletal environments. This review summarizes studies that describe various approaches to improving the therapeutic capability of cell therapies in these harsh environments. The result is an overview of what approaches can be targeted and/or combined to develop a more consistent cell therapy for musculoskeletal pathologies.

The intervertebral disc contributes to motion, weight bearing, and flexibility of the spine, but is susceptible to damage and morphological changes that contribute to pathology with age and injury. Engineering strategies that rely upon synthetic materials or composite implants that do not interface with the biological components of the disc have not met with widespread use or desirable outcomes in the treatment of intervertebral disc pathology. Here we review bioengineering advances to treat disc disorders, using cell-supplemented materials, or acellular, biologically based materials, that provide opportunity for cell-material interactions and remodeling in the treatment of intervertebral disc disorders. While a field still in early development, bioengineering-based strategies employing novel biomaterials are emerging as promising alternatives for clinical treatment of intervertebral disc disorders.

Degenerative disc disease (DDD) is a primary contributor to low-back pain, a leading cause of disability. Progression of DDD is aided by inflammatory cytokines in the intervertebral disc (IVD), particularly TNF-α and IL-1β, but current treatments fail to effectively target this mechanism. The objective of this study was to explore the feasibility of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) epigenome editing-based therapy for DDD, by modulation of TNFR1/IL1R1 signaling in pathological human IVD cells. Human IVD cells from the nucleus pulposus of patients receiving surgery for back pain were obtained and the regulation of TNFR1/IL1R1 signaling by a lentiviral CRISPR epigenome editing system was tested. These cells were tested for successful lentiviral transduction/expression of deactivated Cas9 fused to Kruppel Associated Box system and regulation of TNFR1/IL1R1 expression. TNFR1/IL1R1 signaling disruption was investigated through measurement of NF-κB activity, apoptosis, and anabolic/catabolic changes in gene expression postinflammatory challenge. CRISPR epigenome editing systems were effectively introduced into pathological human IVD cells and significantly downregulated TNFR1 and IL1R1. This downregulation significantly attenuated deleterious TNFR1 signaling but not IL1R1 signaling. This is attributed to less robust IL1R1 expression downregulation, and IL-1β-driven reversal of IL1R1 expression downregulation in a portion of patient IVD cells. In addition, RNAseq data indicated novel transcription factor targets, IRF1 and TFAP2C, as being primary regulators of inflammatory signaling in IVD cells. These results demonstrate the feasibility of CRISPR epigenome editing of inflammatory receptors in pathological IVD cells, but highlight a limitation in epigenome targeting of IL1R1. This method has potential application as a novel gene therapy for DDD, to attenuate the deleterious effect of inflammatory cytokines present in the degenerative IVD.

Musculoskeletal tissues contain critical gradients in extracellular matrix (ECM) composition and cell types that allow for proper mechanical function of tissues and integration between adjacent tissues. However, properly controlling these patterns in engineered tissues is difficult and tissue engineering (TE) is presently in need of methods to generate integration zones for tissue anchoring, transition zones between tissues, and controlled ECM gradients for proper mechanical function. In this study, we present a novel method of using a microfluidic flow cell array (MFCA) to precisely control cell deposition onto TE constructs to produce tunable cell patterns on engineered constructs. In this study, we characterized MFCA cell deposition to efficiently and reliably deposit cells in controllable patterns and densities. We developed methods for deposition of human adipose-derived stem cells and human osteoblasts using a 12-channel pilot printhead. We mimicked key gradients and transitions by creating two-cell and three-cell-type transitions characteristic of the integration zones of musculoskeletal tissues. Overall, we demonstrate the ability to precisely and reproducibly control cell deposition on engineered constructs using this method and control cell population gradients. We establish the production of multicell transitions and multicell interfaces utilizing MFCA cell deposition, to demonstrate the potential of the method to create an extensive variety of engineered musculoskeletal tissues. Furthermore, customization of the printhead design can accommodate various structures, sizes, shapes, and number of flow cell channels to meet specific requirements for a broad range of musculoskeletal tissues.

Background Methods for the detection of the temporal and spatial generation of painful symptoms are needed to improve the diagnosis and treatment of painful neuropathies and to aid preclinical screening of molecular therapeutics. Methods In this study, we utilized in vivo luminescent imaging of NF-κB activity and serum cytokine measures to investigate relationships between the NF-κB regulatory network and the presentation of painful symptoms in a model of neuropathy. Results The chronic constriction injury model led to temporal increases in NF-κB activity that were strongly and non-linearly correlated with the presentation of pain sensitivities (i.e. mechanical allodynia and thermal hyperalgesia). The delivery of NEMO-binding domain peptide reduced pain sensitivities through the inhibition of NF-κB activity in a manner consistent with the demonstrated non-linear relationship. Importantly, the combination of non-invasive measures of NF-κB activity and NF-κB-regulated serum cytokines produced a highly predictive model of both mechanical (R2 = 0.86) and thermal (R2 = 0.76) pain centred on the NF-κB regulatory network (NF-κB, IL-6, CXCL1). Conclusions Using in vivo luminescent imaging of NF-κB activity and serum cytokine measures, this work establishes NF-κB and NF-κB-regulated cytokines as novel multivariate biomarkers of pain-related sensitivity in this model of neuropathy that may be useful for the rapid screening of novel molecular therapeutics.

We developed a new method to manufacture dense, aligned, and porous collagen scaffolds using biaxial plastic compression of type I collagen gels. Using a novel compression apparatus that constricts like an iris diaphragm, low density collagen gels were compressed to yield a permanently densified, highly aligned collagen material. Micro-porosity scaffolds were created using hydrophilic elastomer porogens that can be selectively removed following biaxial compression, with porosity modulated by using different porogen concentrations. The resulting scaffolds exhibit collagen densities that are similar to native connective tissues (∼10% collagen by weight), pronounced collagen alignment across multiple length scales, and an interconnected network of pores, making them highly relevant for use in tissue culture, the study of physiologically relevant cell-matrix interactions, and tissue engineering applications. The scaffolds exhibited highly anisotropic material behavior, with the modulus of the scaffolds in the fiber direction over 100 times greater than the modulus in the transverse direction. Adipose-derived mesenchymal stem cells were seeded onto the biaxially compressed scaffolds with minimal cell death over seven days of culture, along with cell proliferation and migration into the pore spaces. This fabrication method provides new capabilities to manufacture structurally and mechanically relevant cytocompatible scaffolds that will enable more physiologically relevant cell culture studies. Further improvement of manufacturing techniques has the potential to produce engineered scaffolds for direct replacement of dense connective tissues such as meniscus and annulus fibrosus. Statement of Significance In vitro studies of cell-matrix interactions and the engineering of replacement materials for collagenous connective tissues require biocompatible scaffolds that replicate the high collagen density (15–25%/wt), aligned fibrillar organization, and anisotropic mechanical properties of native tissues. However, methods for creating scaffolds with these characteristics are currently lacking. We developed a new apparatus and method to create high density, aligned, and porous collagen scaffolds using a biaxial compression with porogens technique. These scaffolds have a highly directional structure and mechanical properties, with the tensile strength and modulus up to 100 times greater in the direction of alignment. We also demonstrated that the scaffolds are a suitable material for cell culture, promoting cell adhesion, viability, and an aligned cell morphology comparable to the cell morphology observed in native aligned tissues.

Back pain is the leading cause of disability globally and the second most common cause of doctors’ visits. Despite extensive research efforts, the underlying mechanism of back pain has not been fully elucidated. The intervertebral disc (IVD) is a viscoelastic tissue that provides flexibility to the spinal column and acts as a shock absorber in the spine. When viscoelastic materials like the IVD are cyclically loaded, they dissipate energy as heat. Thus, diurnal, regular movements of the vertebral column that deform the IVD could increase disc temperature through viscoelastic heating. This temperature rise has the potential to influence cell function, drive cell death and induce nociception in innervating nociceptive neurons within the IVD. The present study was conducted to investigate the capacity of IVD to increase in temperature due to viscoelastic heating. Insulated caudal bovine IVD were subjected to physiological cyclic uniaxial compression over a range of frequencies (0.1‐15 Hz) and loading durations (1‐10 min) ex vivo, and the temperature rise in the tissue was recorded. According to our findings, the IVD can experience a temperature rise of up to 2.5°C under cyclic loading. Furthermore, under similar conditions, the inner nucleus pulposus exhibits more viscoelastic heating than the outer annulus fibrosis, likely due to its more viscous composition. The measured temperature rise of the disc has physiological relevance as degenerative IVD tissue has been shown to produce a sensitization of nociceptive neurons that spontaneously fire at 37°C, with a T50 response at 37.3°C and a maximum response at 38°C. Our results suggest that viscoelastic heating of IVD could interact with sensitized nociceptive neurons in the degenerative IVD to play a role in back pain.

Musculoskeletal diseases have been associated with inflammatory cytokine action, particularly action by TNF-α and IL-1β. These inflammatory cytokines promote apoptosis and senescence of cells in diseased tissue and extracellular matrix breakdown. Stem cell-based therapies are being considered for the treatment of musculoskeletal diseases, but the presence of these inflammatory cytokines will have similar deleterious action on therapeutic cells delivered to these environments. Methods that prevent inflammatory-induced apoptosis and proinflammatory signaling, in cell and pathway-specific manners are needed. In this study we demonstrate the use of clustered regularly interspaced short palindromic repeats (CRISPR)-based epigenome editing to alter cell response to inflammatory environments by repressing inflammatory cytokine cell receptors, specifically TNFR1 and IL1R1. We targeted CRISPR/Cas9-based repressors to TNFR1 and IL1R1 gene regulatory elements in human adipose-derived stem cells (hADSCs) and investigated the functional outcomes of repression of these genes. Efficient signaling regulation was demonstrated in engineered hADSCs, as activity of the downstream transcription factor NF-κB was significantly reduced or maintained at baseline levels in the presence of TNF-α or IL-1β. Pellet culture of undifferentiated hADSCs demonstrated improved survival in engineered hADSCs treated with TNF-α or IL-1β, while having little effect on their immunomodulatory properties. Furthermore, engineered hADSCs demonstrated improved chondrogenic differentiation capacity in the presence of TNF-α or IL-1β, as shown by superior production of glycosaminglycans in this inflammatory environment. Overall this work demonstrates a novel method for modulating cell response to inflammatory signaling that has applications in engineering cells delivered to inflammatory environments, and as a direct gene therapy to protect endogenous cells exposed to chronic inflammation, as observed in a broad spectrum of degenerative musculoskeletal pathology.

Back pain is a major contributor to disability and has significant socioeconomic impacts worldwide. The degenerative intervertebral disc (IVD) has been hypothesized to contribute to back pain, but a better understanding of the interactions between the degenerative IVD and nociceptive neurons innervating the disc and treatment strategies that directly target these interactions is needed to improve our understanding and treatment of back pain. We investigated degenerative IVD-induced changes to dorsal root ganglion (DRG) neuron activity and utilized CRISPR epigenome editing as a neuromodulation strategy. By exposing DRG neurons to degenerative IVD-conditioned media under both normal and pathological IVD pH levels, we demonstrate that degenerative IVDs trigger interleukin (IL)-6-induced increases in neuron activity to thermal stimuli, which is directly mediated by AKAP and enhanced by acidic pH. Utilizing this novel information on AKAP-mediated increases in nociceptive neuron activity, we developed lentiviral CRISPR epigenome editing vectors that modulate endogenous expression of AKAP150 by targeted promoter histone methylation. When delivered to DRG neurons, these epigenome-modifying vectors abolished degenerative IVD-induced DRG-elevated neuron activity while preserving non-pathologic neuron activity. This work elucidates the potential for CRISPR epigenome editing as a targeted gene-based pain neuromodulation strategy.

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.

Objective To investigate the relationship between NF-κB activity, cytokine levels, and pain sensitivities in a rodent model of osteoarthritis (OA).

Cell delivery to the pathological intervertebral disc (IVD) has significant therapeutic potential for enhancing IVD regeneration. The development of injectable biomaterials that retain delivered cells, promote cell survival, and maintain or promote an NP cell phenotype in vivo remains a significant challenge. Previous studies have demonstrated NP cell – laminin interactions in the nucleus pulposus (NP) region of the IVD that promote cell attachment and biosynthesis. These findings suggest that incorporating laminin ligands into carriers for cell delivery may be beneficial for promoting NP cell survival and phenotype. Here, an injectable, laminin-111 functionalized poly(ethylene glycol) (PEG-LM111) hydrogel was developed as a biomaterial carrier for cell delivery to the IVD. We evaluated the mechanical properties of the PEG-LM111 hydrogel, and its ability to retain delivered cells in the IVD space. Gelation occurred in approximately 20 minutes without an initiator, with dynamic shear moduli in the range of 0.9 – 1.4 kPa. Primary NP cell retention in cultured IVD explants was significantly higher over 14 days when cells were delivered within a PEG-LM111 carrier, as compared to cells in liquid suspension. Together, these results suggest this injectable laminin-functionalized biomaterial may be an easy to use carrier for delivering cells to the IVD.

Genome and epigenome editing have emerged as convenient and effective tools for scientific research in a broad spectrum of fields. Recent advances in genome and epigenome editing tools have made these systems more accessible, more efficient, and precise. In this chapter, we will describe the mechanisms of the primary genome and epigenome editing strategies and explore their mechanisms of action, with a focus on ZFN, TALEN, CRISPR/Cas9, and CRISPR/dCas9 systems, and highlight their application to therapeutic development and biomaterials research.

The current tools available to physicians for the treatment of back pain are relatively limited, palliative in nature, and often lead to deleterious side effects. As a result, much research is occurring in the biomaterials realm to create new treatments that not only relieve pain, but restore function to the spine and circumvent the unwanted side effects. Both traditional metal/polyethylene hardware approaches, often associated with joint replacements, and more novel regenerative medicine approaches are being investigated. It is currently unclear which procedures and materials will be best suited for the treatment of back pain, but a wide range of materials are being investigated. This chapter reviews the current state of biomaterial-based treatment of intervertebral disc-originated back pain. Topics cover established treatments such as spinal fusion and total disc replacement, as well as novel regenerative treatments such as nucleus pulposus replacement, annulus fibrosus repair, and tissue-engineered total disc replacement.

Objective Osteoarthritis (OA) is a consequence of not only mechanical events such as joint instability, but also biological events that result in the upregulation of proinflammatory and catabolic mediators. The intra-articular injection of monoiodoacetate (MIA) has been widely used to induce OA. NF-κB activity has been linked to increased expression of proinflammatory cytokines (IL-1β, TNF-α, IL-6, etc), metalloproteinases (MMPs), chemokines and inducible enzymes, which all contribute to cartilage degradation and subsequent OA. The goal of this study was to use in vivo imaging (IVIS) of NF-κB activation to track longitudinal changes due to inflammation in a rodent model of OA. Design Twenty-four (24) NF-κB-luc reporter transgenic mice [BALB/C-Tg (NF-κB-RE-luc)-Xen, age 7-8 weeks] were given intra-articular knee injections with either MIA (n = 12) or normal saline (n = 12) to serve as a control. IVIS and ex vivo imaging of NF-κB and tactile allodynia measurements were performed, and correlations were recorded preoperatively and on days 1, 3, 7, 14, 21 and 28. Animals were euthanized on days 3 and 28 for ex vivo imaging, and tissues were stored for future immunohistochemical evaluation. Results NF-κB activity was significantly elevated in the MIA group on days 1 and 3 (p < 0.05) when compared to preoperative levels and was significantly elevated compared to the normal saline group on day 3 (p < 0.05). There was a significant increase in tactile allodynia in the MIA group compared to preoperative levels, as well as compared to the normal saline group at all time points (p < 0.05). In vivo NF-κB luminescence correlated with tactile allodynia (p < 0.0001) and with ex vivo imaging (p < 0.0001). Conclusion This study validates the use of IVIS imaging of NF-κB activity in a MIA rodent model of arthritis and provides evidence for the use of NF-κB luminescence imaging as an imaging biomarker of pain sensitivities. This can be utilized in the future to further elucidate NF-κB's role in inflammation and OA. In addition, it can help evaluate potential therapeutic agents that target NF-κB. Mata BA, Bowles R, Mwangi TK, Setton LA. Impaired Function and in vivo Imaging of NF-κB Activation in a Mouse Model of Knee Joint Inflammation. The Duke Orthop J 2013;3(1):41-47.

Because of cost and time, it is difficult to relate to students how fundamental chemical principles are involved in cutting edge biomedical breakthroughs being reported in the national media. The laboratory exercise presented here is aimed at high school chemistry students and uses alginate hydrogels, a common material used in tissue engineering, to help students explore the relationship between chemical bonding and material properties while relating it to the field of tissue engineering. In addition, this lab is designed as a model based inquiry exercise to provide a better understanding of how contemporary science is practiced. The lab is intended to be used as part of a four day curriculum on tissue engineering but can be done together with the supporting curriculum or separately. The exercise is inexpensive, approximately $6.00 per student group and can be performed in low-resource laboratories, as it requires no elaborate equipment. The students who completed these exercises showed enhanced understanding of ionic bonding and were able to describe how such bonding related to the properties of materials.

Lower back and neck pain are leading physical conditions for which patients see their doctors in the United States. The organ commonly implicated in this condition is the intervertebral disc (IVD), which frequently herniates, ruptures, or tears, often causing pain and limiting spinal mobility. To date, approaches for replacement of diseased IVD have been confined to purely mechanical devices designed to either eliminate or enable flexibility of the diseased motion segment. Here we present the evaluation of a living, tissue-engineered IVD composed of a gelatinous nucleus pulposus surrounded by an aligned collagenous annulus fibrosus in the caudal spine of athymic rats for up to 6 mo. When implanted into the rat caudal spine, tissue-engineered IVD maintained disc space height, produced de novo extracellular matrix, and integrated into the spine, yielding an intact motion segment with dynamic mechanical properties similar to that of native IVD. These studies demonstrate the feasibility of engineering a functional spinal motion segment and represent a critical step in developing biological therapies for degenerative disc disease.

Nonbiological total disc replacement is currently being used for the treatment of intervertebral disc (IVD) disease and injury, but these implants are prone to mechanical wear, tear and possible dislodgement. Recently, tissue-engineered total disc replacement (TE-TDR) has been investigated as a possible alternative to more fully replicate the native IVD properties. However, the performance of TE-TDRs has not been studied in the native disc space. In this study, MRI and microcomputed tomography imaging of the rat spine were used to design a collagen (annulus fibrosus)/alginate (nucleus pulposus) TE-TDR to a high degree of geometric accuracy, with less than 10% difference between TE-TDR and the native disc dimensions. Image-based TE-TDR implants were then inserted into the L4/L5 disc space of athymic rats (n = 5) and maintained for 16 weeks. The disc space was fully or partially maintained in three of five animals and proteoglycan and collagen histology staining was similar in composition to the native disc. In addition, good integration was observed between TE-TDR and the vertebral bodies, as well as remnant native IVD tissue. Overall, this study provides evidence that TE-TDR strategies may yield a clinically viable treatment for diseased or injured IVD.

Study design: Prospective randomized animal study. Objective: To determine a surgical technique for reproducible and functional intervertebral disc replacement in an orthotopic animal model. Methods: The caudal 3/4 intervertebral disc (IVD) of the rat tail was approached by two surgical techniques: blunt dissection, stripping and retracting (Technique 1) or incising and repairing (Technique 2) the dorsal longitudinal tendons. The intervertebral disc was dissected and removed, and then either discarded or reinserted. Outcome measures were perioperative complications, spontaneous tail movement, 7T MRI (T1- and T2-sequences for measurement of disc space height (DSH) and disc hydration). Microcomputed tomographic imaging (micro CT) was additionally performed postmortem. Results: No vascular injuries occurred and no systemic or local infections were observed over the course of 1 month. Tail movements were maintained. With tendon retraction (Technique 1) gross loss of DSH occurred with both discectomy and reinsertion. Tendon division (Technique 2) maintained DSH with IVD reinsertion but not without. The DSH was demonstrated on MRI measurement. A new scoring system to assess IVD appearances was described. Conclusions: The rat tail model, with a tendon dividing surgical technique, can function as an orthotopic animal model for IVD research. Mechanical stimulation is maintained by preserved tail movements. 7T MRI is a feasible modality for longitudinal monitoring for the rat caudal disc.

Study type: Basic science Introduction: Chronic back pain due to degenerative disc disease (DDD) is among the most important medical conditions causing morbidity and significant health care costs. Surgical treatment options include disc replacement or fusion surgery, but are associated with significant short- and long-term risks.1 Biological tissue-engineering of human intervertebral discs (IVD) could offer an important alternative.2 Recent in vitro data from our group have shown successful engineering and growth of ovine intervertebral disc composites with circumferentially aligned collagen fibrils in the annulus fibrosus (AF) (Figure 1).3 Figure 1 Tissue-engineered composite disc a Experimental steps to generate composite tissue-engineered IVDs3 b Example of different AF formulations on collagen alignment in the AF. Second harmonic generation and two-photon excited fluorescence images of seeded collagen gels (for AF) of 1 and 2.5 mg/ml over time. At seeding, cells and collagen were homogenously distributed in the gels. Over time, AF cells elongated and collagen aligned parallel to cells. Less contraction and less alignment is noted after 3 days in the 2.5 mg/mL gel. c Imaging-based creation of a virtual disc model that will serve as template for the engineered disc. Total disc dimensions (AF and NP) were retrieved from micro-computer tomography (CT) (left images), and nucleus pulposus (NP) dimensions alone were retrieved from T2-weighted MRI images (right images). Merging of MRI and micro-CT models revealed a composite disc model (middle image)—Software: Microview, GE Healthcare Inc., Princeton, NJ; and slicOmatic v4.3, TomoVision, Montreal, Canada. d Flow chart describing the process for generating multi-lamellar tissue engineered IVDs. IVDs are produced by allowing cell-seeded collagen layers to contract around a cell-seeded alginate core (NP) over time Objective: The next step is to investigate if biological disc implants survive, integrate, and restore function to the spine in vivo. A model will be developed that allows efficient in vivo testing of tissue-engineered discs of various compositions and characteristics. Methods: Athymic rats were anesthetized and a dorsal approach was chosen to perform a microsurgical discectomy in the rat caudal spine (Fig. 2,Fig. 3). Control group I (n = 6) underwent discectomy only, Control group II (n = 6) underwent discectomy, followed by reimplantation of the autologous disc. Two treatment groups (group III, n = 6, 1 month survival; group IV, n = 6, 6 months survival) received a tissue-engineered composite disc implant. The rodents were followed clinically for signs of infection, pain level and wound healing. X-rays and magnetic resonance imaging (MRI) were assessed postoperatively and up to 6 months after surgery (Fig. 6,Fig. 7). A 7 Tesla MRI (Bruker) was implemented for assessment of the operated level as well as the adjacent disc (hydration). T2-weighted sequences were interpreted by a semiquantitative score (0 = no signal, 1 = weak signal, 2 = strong signal and anatomical features of a normal disc). Histology was performed with staining for proteoglycans (Alcian blue) and collagen (Picrosirius red) (Fig. 4,Fig. 5). Figure 2 Disc replacement surgery a Operative situs with native disc that has been disassociated from both adjacent vertebrae b Native disc (left) and tissue-engineered implant (right) c Implant in situ before wound closureAF: Annulus fi brosus, nP: nucleus pulposus, eP: endplate, M: Muscle, T: Tendon, s: skin, art: artery, GP: Growth plate, B: Bone Figure 3 Disc replacement surgery. Anatomy of the rat caudal disc space a Pircrosirius red stained axial cut of native disc space b Saffranin-O stained sagittal cut of native disc space Figure 4 Histologies of three separate motion segments from three different rats. Animal one = native IVD, Animal two = status after discectomy, Animal three = tissue-engineered implant (1 month) a–c H&E (overall tissue staining for light micrsocopy) d–f Alcian blue (proteoglycans) g–i Picrosirius red (collagen I and II) Figure 5 Histology from one motion segment four months after implantation of a bio-engineered disc construct a Picrosirius red staining (collagen) b Polarized light microscopy showing collagen staining and collagen organization in AF region c Increased Safranin-O staining (proteoglycans) in NP region of the disc implant d Higher magnification of figure 5c: Integration between implanted tissue-engineered total disc replacement and vertebral body bone Figure 6 MRI a Disc space height measurements in flash/T1 sequence (top: implant (714.0 micrometer), bottom: native disc (823.5 micrometer) b T2 sequence, red circle surrounding the implant NP Figure 7 7 Tesla MRI imaging of rat tail IVDs showing axial images (preliminary pilot data) a Diffusion tensor imaging (DTI) on two explanted rat tail discs in Formalin b Higher magnification of a, showing directional alignment of collagen fibers (red and green) when compared to the color ball on top which maps fibers' directional alignment (eg, fibers directing from left to right: red, from top to bottom: blue) c Native IVD in vivo (successful imaging of top and bottom of the IVD (red) d Gradient echo sequence (GE) showing differentiation between NP (light grey) and AF (dark margin) e GE of reimplanted tail IVD at the explantation level f T1Rho sequence demonstrating the NP (grey) within the AF (dark margin), containing the yellow marked region of interest for value acquisition (preliminary data are consistent with values reported in the literature). g T2 image of native IVD in vivo for monitoring of hydration (white: NP) Results: The model allowed reproducible and complete discectomies as well as disc implantation in the rat tail spine without any surgical or postoperative complications. Discectomy resulted in immediate collapse of the disc space. Preliminary results indicate that disc space height was maintained after disc implantation in groups II, III and IV over time. MRI revealed high resolution images of normal intervertebral discs in vivo. Eight out of twelve animals (groups III and IV) showed a positive signal in T2-weighted images after 1 month (grade 0 = 4, grade 1 = 4, grade 2 = 4). Positive staining was seen for collagen as well as proteoglycans at the site of disc implantation after 1 month in each of the six animals with engineered implants (group III). Analysis of group IV showed positive T2 signal in five out of six animals and disc-height preservation in all animals after 6 months. Conclusions: This study demonstrates for the first time that tissue-engineered composite IVDs with circumferentially aligned collagen fibrils survive and integrate with surrounding vertebral bodies when placed in the rat spine for up to 6 months. Tissue-engineered composite IVDs restored function to the rat spine as indicated by maintenance of disc height and vertebral alignment. A significant finding was that maintenance of the composite structure in group III was observed, with increased proteoglycan staining in the nucleus pulposus region (Figure 4d–f). Proteoglycan and collagen matrix as well as disc height preservation and positive T2 signals in MRI are promising parameters and indicate functionality of the implants.

Many cartilaginous tissues such as intervertebral disc (IVD) display a heterogeneous collagen microstructure that results in mechanical anisotropy. These structures are responsible for mechanical function of the tissue and regulate cellular interactions and metabolic responses of cells embedded within these tissues. Using collagen gels seeded with ovine annulus fibrosus cells, constructs of varying structure and heterogeneity were created to mimic the circumferential alignment of the IVD. Alignment was induced within gels by contracting annular gels around an inner boundary using both a polyethylene center and alginate center to create a composite engineered IVD. Collagen alignment and heterogeneity were measured using second harmonic generation microscopy. Decreasing initial collagen density from 2.5 mg/mL to 1 mg/mL produced greater contraction of constructs, resulting in gels that were 55% and 6.2% of the original area after culture, respectively. As a result, more alignment occurred in annular-shaped 1 mg/mL gels compared with 2.5 mg/mL gels (p

To determine the utility of silk fibroin (SF) microparticles as sustained release vehicles for intra-articular delivery.SF formulations were varied to generate microparticle drug carriers that were characterized in vitro for their physical properties, release kinetics for a conjugated fluorophore (Cy7), and in vivo for intra-articular retention time using live-animal, fluorescence in vivo imaging.SF microparticle carriers were spherical in shape and ranged from 598 nm to 21.5 μm in diameter. SF microparticles provided for sustained release of Cy7 in vitro, with only 10% of the initial load released over 7 days. Upon intra-articular injection in rat knee joints, the SF microparticles were associated with an intra-articular fluorescence decay half-life of 43.3h, greatly increasing the joint residence over that for an equivalent concentration of SF-Cy7 in solution form. The SF microparticles also increase the localization of dye within the joint cavity as determined by image analysis of fluorescent gradients, significantly reducing distribution of the Cy7 to neighboring tissue as compared to SF-Cy7 in free solution.Silk microparticles act to provide for localized and sustained delivery of loaded small molecules following intra-articular injection, and may be an attractive strategy for delivering small molecule drugs for the treatment of arthritis.

The outcome of the complex process of bone regeneration is the restoration of form and function, ideally, enduring, to a bone insufficiency. This chapter attempts to answer the question of how to harness the intrinsic regenerative capacity of bone? The approach to bone regeneration for this chapter is to highlight bone regeneration by exploiting contemporary, albeit pedestrian approaches of autografts, allografts and xenografts. First, the authors will define these modalities and emphasize what is good about them and what is lacking. Consensus definitions of the biological and biomechanical properties of regeneration, stating specific performance parameters that define the temporal road map for the regenerative cascade will be offered. A fracture healing model as a prototype for the regenerative process will be exploited. Key directional concepts for the rational design and development of a regenerative therapy will be introduced. These concepts will include the osteogenic biohemodynamic cascade and for bone regenerative therapies, the importance of a 4D environmental blastema matrix where embryogenesis is recapitulated. Finally, stringent performance criteria will be offered with objective data as a framework for the rational design and fielding of a master tool kit for bone regenerative therapeutics.

To investigate the relationship between NF-κB activity, cytokine levels, and pain sensitivities in a rodent model of osteoarthritis (OA).OA was induced in transgenic NF-κB-luciferase reporter mice via intraarticular injection of monosodium iodoacetate (MIA). Using luminescence imaging we evaluated the temporal kinetics of NF-κB activity and its relationship to the development of pain sensitivities and serum cytokine levels in this model.MIA induced a transient increase in joint-related NF-κB activity at early time points (day 3 after injection) and an associated biphasic pain response (mechanical allodynia). NF-κB activity, serum interleukin-6 (IL-6), IL-1β, and IL-10 levels accounted for ∼75% of the variability in pain-related mechanical sensitivities in this model. Specifically, NF-κB activity was strongly correlated with mechanical allodynia and serum IL-6 levels in the inflammatory pain phase of this model (day 3), while serum IL-1β was strongly correlated with pain sensitivities in the chronic pain phase of the model (day 28).Our findings suggest that NF-κB activity, IL-6, and IL-1β may play distinct roles in pain sensitivity development in this model of arthritis and may distinguish the acute pain phase from the chronic pain phase. This study establishes luminescence imaging of NF-κB activity as a novel imaging biomarker of pain sensitivities in this model of OA.

Biological and anatomical changes of intervertebral disc (IVD) degeneration frequently occur in the nucleus pulposus (NP) [1]. Changes in NP matrix composition coincide with the loss of a distinct notochord derived cell population [2],[3], which may have the potential to generate or maintain a functional NP-like matrix. Immature NP cells reside in an environment rich in laminin and express specific laminin-binding receptors [4],[5]. Additionally, NP cells attach in higher numbers to laminins as compared to cells isolated from other regions of the IVD [6]. Our initial work demonstrated that matrix protein and stiffness modulate NP cell-cell interactions upon surfaces [7], with results that suggest soft, laminin-functionalized hydrogels may be useful for promoting an NP-like cell phenotype.Copyright © 2012 by ASME

Nonbiological total disc replacement is currently being used for the treatment of intervertebral disc (IVD) disease and injury, but these implants are prone to mechanical wear, tear and possible dislodgement. Recently, tissue-engineered total disc replacement (TE-TDR) has been investigated as a possible alternative to more fully replicate the native IVD properties. However, the performance of TE-TDRs has not been studied in the native disc space. In this study, MRI and microcomputed tomography imaging of the rat spine were used to design a collagen (annulus fibrosus)/alginate (nucleus pulposus) TE-TDR to a high degree of geometric accuracy, with less than 10% difference between TE-TDR and the native disc dimensions. Image-based TE-TDR implants were then inserted into the L4/L5 disc space of athymic rats (n = 5) and maintained for 16 weeks. The disc space was fully or partially maintained in three of five animals and proteoglycan and collagen histology staining was similar in composition to the native disc. In addition, good integration was observed between TE-TDR and the vertebral bodies, as well as remnant native IVD tissue. Overall, this study provides evidence that TE-TDR strategies may yield a clinically viable treatment for diseased or injured IVD.

Selected abstracts delivered at the 9th Annual AOSpine North America Fellows Forum Consistent with EBSJ's commitment to fostering quality research, we are pleased to feature some of the most highly rated abstracts from the 9th Annual AOSpine North America Fellows Forum in Banff, Canada. Enhancing the quality of evidence in spine care means acknowledging and supporting the efforts of young researchers within our AOSpine North America network. We look forward to seeing more from these promising researchers in the future.

Herniation of the intervertebral disc may be associated with a painful neuropathy (sciatica) or motor weakness that is partly mediated by inflammatory cell infiltration and proinflammatory cytokine expression. Much of the cytokine-mediated signaling and inflammatory events falls downstream of activation of the transcription factor, NF- κB. We have previously demonstrated an ability to visualize NF- κB activity in a transgenic NF-κB luciferase reporter mouse, following induction of arthritis in the knee joint.1 The temporal and spatial pattern of NF-κB-luc correlated with development of painful sensitivities in the arthritis model suggesting an ability to use in vivo imaging to rapidly screen progression of pathology and therapeutic interventions. Here, we investigate the temporal and spatial development of NF-κB activity in the NF-κB-luc reporter mouse following induction of peripheral nerve injury (sciatic nerve constriction) and test for relationships amongst pain-related sensitivities, serum levels of NF-κB-related cytokines, and regional measures of NF-κB activity. Our work in this model system revealed that NF-κB B activity following nerve injury is elevated in the region of the affected nerve, with luciferase production specifically from the sciatic nerve and the surrounding musculature. Temporal elevations in NF-κB activity were noted beginning at 3 days after surgery, and correlated with mechanical (von Frey testing, Spearman ρ-0.84) and thermal (hyperalgesia at paw, Spearman ρ -0.80) sensitivity in a distinctly nonlinear fashion. NF- κB activity also correlated with serum interleukin 6 levels (Spearman ρ 0.75), but not with other measured cytokines. Delivery of the NF- κB specific inhibitor, NEMO-binding domain peptide NBD, was able to reduce NF- κB-luc signal, restore thermal sensitivity to sham levels, and restore mechanical sensitivity to near sham levels. These findings are important for demonstrating a direct link between the NF- κB-luc signal and pain-related measures. Together this work suggests that longitudinal and spatially resolved in vivo imaging can be useful to characterize cytokine-related sensitivities in neuropathy, with implications for radiculopathy associated with intervertebral disc herniation. Disclosure of Interest None declared Reference Bowles RD, Mata BA, Bell RD, et al. In vivo luminescent imaging of NF-κB activity and serum cytokine levels predict pain sensitivities in a rodent model of osteoarthritis. Arthritis Rheum 2013; November 18 (Epub ahead of print); doi: 10.1002/art.38279

Extended abstract of a paper presented at Microscopy and Microanalysis 2010 in Portland, Oregon, USA, August 1 – August 5, 2010.

Introduction Degenerative disk disease is a leading cause of disability in the developed countries and over the past few decades, has become a major public health problem worldwide due to the significant improvement of life expectancy. Current medical and surgical treatment strategies for DDD mainly focus on pain and symptom relief, and do not necessarily address the underlying pathology of the disease. In recent years, there has been an increasing interest in developing biological based implants as an alternative, which could restore function and mobility of the spine. Here, we present the preliminary results of our study, in which five different compositions of tissue-engineered intervertebral disks were implanted into the rat caudal spine, and radiographic studies were used to evaluate the results. Materials and Methods Based on MRI and micro-computed tomography imaging of the rat spine, compound tissue-engineered total disk replacement implants (TE-TDR) were designed to a high degree of geometric accuracy. TE-TDRs were composed of a gelatinous nucleus pulposus (NP) surrounded by an aligned collagenous annulus fibrosus. Five different compositions of the TE-TDR were designed, containing different concentrations of collagen in the AF and different concentrations of sheep chondrocytes seeded in their NP. TE-TDR implants were then inserted into the S3/S4 caudal disk space of 37 mature athymic nude rats (RNU) and were maintained on average for 15 weeks. In group 1 (four rats), TE-TDRs contained 10 million sheep cells/mL in their NP and 1 mg/mL collagen in their AF; in a similar way group 2 (four rats) received TE-TDRs with 10 million cells/mL NP and 2 mg/mL collagen AF; group 3 (eight rats) received TE-TDRs with 1 million cells/mL NP and 2 mg/mL collagen AF; in groups 4 and 5, the TE-TDRs' cell and collagen concentrations were similar to those of group 3, however in group 4 (14 rats), the disks were additionally photo cross-linked and cultured in riboflavin within their development process; and in group 5 (seven rats), an additional layer of collagen gel was pipetted around each disk to produce a second lamella. X-rays were performed both after surgery and at the latest follow-up time. Disk height changes were evaluated by calculating the Disk Height Indices (DHI) on X-rays. Based on the rostral healthy disk, a baseline DHI was also calculated for each animal. The results were then presented as percentages, comparing the disk height of the level of interest to the rostral healthy disk height. Statistical analyses were performed comparing the implanted rats with a control group in the previously published data; comparisons were also been made between the different groups of implanted rats. Results Five different compositions of the TE-TDR with differences in their collagen and cell contents were designed and implanted in the S3/S4 caudal disk space of 37 rats; The radiographic results of these five groups were then compared to the previously published data on a control group (six rats), on which simple discectomies were performed. Both after the operation and at the latest follow-up, the mean disk height in implanted animals was significantly higher than the control group (51% and 61% compared to 37%). Interestingly, the disk height in the implanted group at the latest follow-up also showed a significant increase, compared to the postoperative disk height. Comparisons between different implanted groups, showed that group 3 (1 million cells/mL and 2 mg/mL collagen) was associated with best results both postoperatively and at the latest follow-up; also, the worst outcome were observed in group 5 (multilamellar) postoperatively, and in group 4 (cross-linked) at latest follow-up; nonetheless no statistical significance was detected. Conclusion The findings of the present study, while preliminary, suggest that TE-TDR may yield a clinically feasible and efficacious treatment for intervertebral disk disease. Although limited by the sample size, this research will serve as a base for further future studies on biological disk replacement. I confirm having declared any potential conflict of interest for all authors listed on this abstract Yes Disclosure of Interest None declared