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Low back pain (LBP) is often associated with the degeneration of human intervertebral discs (IVDs). However, the pain-inducing mechanism in degenerating discs remains to be elucidated. Here, we identified a subtype of locally residing nucleus pulposus cells (NPCs), generated by the environmental stress in degenerating discs, that triggered the onset of discogenic LBP. Single-cell transcriptomic analysis of human tissues showed a strong correlation between this specific pain-triggering subtype and the pain conditions in human degenerated discs. Next, we recreated this pain-triggering subtype by applying known exogenous stressors to healthy NPCsin vitro. The recreated pain phenotype activated functional sensory neurons responsein vitroand induced local inflammatory responses, hyperalgesia, and mechanical sensitivity in a healthy rat IVDin vivo. Our findings provide strong evidence of a previously unknown pain-inducing mechanism mediated by NPCs in degenerating IVDs. This newly defined pathway will aid in the development of NPC-targeted therapeutic strategies for clinically unmet need to attenuate discogenic LBP.One Sentence SummaryDiscogenic low back pain can be initiated by a stress-induced subtype of nucleus pulposus cells present in human degenerating intervertebral discs

Replacement of lost cranial bone (partly mesodermal and partly neural crest‐derived) is challenging and includes the use of nonviable allografts. To revitalize allografts, bone marrow‐derived mesenchymal stromal cells (mesoderm‐derived BM‐MSCs) have been used with limited success. We hypothesize that coating of allografts with induced neural crest cell‐mesenchymal progenitor cells (iNCC‐MPCs) improves implant‐to‐bone integration in mouse cranial defects. Human induced pluripotent stem cells were reprogramed from dermal fibroblasts, differentiated to iNCCs and then to iNCC‐MPCs. BM‐MSCs were used as reference. Cells were labeled with luciferase (Luc2) and characterized for MSC consensus markers expression, differentiation, and risk of cellular transformation. A calvarial defect was created in non‐obese diabetic/severe combined immunodeficiency (NOD/SCID) mice and allografts were implanted, with or without cell coating. Bioluminescence imaging (BLI), microcomputed tomography (μCT), histology, immunofluorescence, and biomechanical tests were performed. Characterization of iNCC‐MPC‐Luc2 vs BM‐MSC‐Luc2 showed no difference in MSC markers expression and differentiation in vitro. In vivo, BLI indicated survival of both cell types for at least 8 weeks. At week 8, μCT analysis showed enhanced structural parameters in the iNCC‐MPC‐Luc2 group and increased bone volume in the BM‐MSC‐Luc2 group compared to controls. Histology demonstrated improved integration of iNCC‐MPC‐Luc2 allografts compared to BM‐MSC‐Luc2 group and controls. Human osteocalcin and collagen type 1 were detected at the allograft‐host interphase in cell‐seeded groups. The iNCC‐MPC‐Luc2 group also demonstrated improved biomechanical properties compared to BM‐MSC‐Luc2 implants and cell‐free controls. Our results show an improved integration of iNCC‐MPC‐Luc2‐coated allografts compared to BM‐MSC‐Luc2 and controls, suggesting the use of iNCC‐MPCs as potential cell source for cranial bone repair., Bone marrow‐derived mesenchymal stromal cells (BM‐MSCs) were obtained from human bone marrow. Induced neural crest cell‐mesenchymal progenitor cells (iNCC‐MPCs) were generated from induced pluripotent stem cell‐derived iNCCs. Cells were luciferase transfected and seeded onto decellularized allografts, which were implanted into 5 mm circular calvarial defects in non‐obese diabetic/severe combined immunodeficiency (NOD/SCID) mice. Allograft only was used as controls. Mice were followed for a duration of 8 weeks. Cell survival, allograft integration, and tissue morphology and transplanted cell contribution were monitored via bioluminescence imaging (BLI), micro‐computed tomography (μCT), and histology and immunofluorescence (IF). Green arrow: BLI; yellow arrow: μCT; Orange arrow: histology and IF; Blue arrow: biomechanical test.

Background Cranial bone loss presents a major clinical challenge and new regenerative approaches to address craniofacial reconstruction are in great demand. Induced pluripotent stem cell (iPSC) differentiation is a powerful tool to generate mesenchymal stem cells (MSCs). Prior research demonstrated the potential of bone marrow-derived MSCs (BM-MSCs) and iPSC-dervied mesenchymal progenitor cells via the neural crest (NCC-MPCs) or mesodermal lineages (iMSCs) to be a promising cell source for bone regeneration. Overexpression of human recombinant bone morphogenetic protein (BMP)6 efficiently stimulates bone formation. The goal of this study was to evaluate the potential of iPSC-derived stem cells via neural crest or mesoderm overexpressing BMP6 and embedded in 3D printable bio-ink to generate viable bone graft alternatives for cranial reconstruction. Methods Cell viability and osteogenic potential of cells and bio-ink (Ink-Bone or GelXa) combinations were investigated in vitro using bioluminescent imaging. Osteogenic potential of bio-ink-cell constructs was evaluated in osteogenic media or nucleofected with BMP6 using qRT-PCR and in vitro µCT. For in vivo testing, two 2mm circular defects were created in the frontal and parietal bones of NOD/SCID mice and treated with Ink-Bone, Ink-Bone + BM-MSC-BMP6, Ink-Bone + iMSC-BMP6 or Ink-Bone + iNCC-MPC-BMP6 or left untreated. For follow up, µCT was performed at weeks 0, 4 and 8 weeks. At time of (week 8), histological and immunofluorescent analyses were performed. Results Both bio-inks supported cell survival and promoted osteogenic differentiation of iNCC-MPCs and BM-MSCs in vitro. At 4 weeks, cell viability of both BM-MSCs and iNCC-MPCs were increased in Ink-Bone compared to GelXA. Combination of Ink-Bone with iNCC-MPC-BMP6 resulted in an increased bone volume in the frontal bone compared to the other groups at 4 weeks post-surgery. At 8 weeks, both iNCC-MPC-BMP6 and iMSC-MSC-BMP6 resulted in an increased bone volume and partial bone bridging between implant and host bone compared to the other groups. Conclusions The results of this study show the potential of NCC-MPC-incorporated bio-ink to regenerate frontal cranial defects. Therefore, this bio-ink-cell combination should be further investigated for its therapeutic potential in large animal models with larger cranial defects, allowing for 3D printing of the cell-incorporated material.

Massive craniofacial bone loss poses a clinical challenge to maxillofacial surgeons. Structural bone allografts are readily available at tissue banks but are rarely used due to a high failure rate. Previous studies showed that intermittent administration of recombinant parathyroid hormone (rPTH) enhanced integration of allografts in a murine model of calvarial bone defect. To evaluate its translational potential, the hypothesis that rPTH would enhance healing of a mandibular allograft in a clinically relevant large animal model of mandibulectomy was tested. Porcine bone allografts were implanted into a 5-cm-long continuous mandible bone defect in six adult Yucatan minipigs, which were randomized to daily intramuscular injections of rPTH (1.75 μg/kg) and placebo (n = 3). Blood tests were performed on Day 56 preoperation, Day 0 and on Day 56 postoperation. Eight weeks after the surgery, bone healing was analyzed using high-resolution X-ray imaging (Faxitron and micro computed tomography [CT]) and three-point bending biomechanical testing. The results showed a significant 2.6-fold rPTH-induced increase in bone formation (p = 0.02). Biomechanically, the yield failure properties of the healed mandibles were significantly higher in the rPTH group (yield load: p < 0.05; energy to yield: p < 0.01), and the post-yield displacement and energy were higher in the placebo group (p < 0.05), suggesting increased mineralized integration of the allograft in the rPTH group. In contrast to similar rPTH therapy studies in dogs, no signs of hypercalcemia, hyperphosphatemia, or inflammation were detected. Taken together, we provide initial evidence that rPTH treatment enhances mandibular allograft healing in a clinically relevant large animal model.

Introduction: As many as 80% of the adult population experience back pain at some point in their lifetimes. Previous studies have indicated a link between back pain and intervertebral disc (IVD) degeneration. Despite decades of research, there is an urgent need for robust stem cell therapy targeting underlying causes rather than symptoms. It has been proposed that notochordal cells (NCs) appear to be the ideal cell type to regenerate the IVD: these cells disappear in humans as they mature, are replaced by nucleus pulposus (NP) cells, and their disappearance correlates with the initiation of degeneration of the disc. Human NCs are in short supply, thus here aimed for generation of notochordal-like cells from induced pluripotent cells (iPSCs). Methods: Human iPSCs were generated from normal dermal fibroblasts by transfecting plasmids encoding for six factors: OCT4, SOX2, KLF4, L-MYC, LIN28, and p53 shRNA. Then the iPSCs were treated with GSK3i to induce differentiation towards Primitive Streak Mesoderm (PSM). The differentiation was confirmed by qRT-PCR and immunofluorescence. PSM cells were transfected with Brachyury (Br)-encoding plasmid and the cells were encapsulated in Tetronic-tetraacrylate-fibrinogen (TF) hydrogel that mimics the NP environment (G'=1kPa), cultured in hypoxic conditions (2% O2) and with specifically defined growth media. The cells were also tested in vivo in a large animal model. IVD degeneration was induced after an annular puncture in pigs, 4 weeks later the cells were injected and IVDs were analyzed at 12 weeks after the injury using MRI, gene expression analysis and histology. Results: After short-term exposure of iPSCs to GSK3i there was a significant change in cell morphology, Primitive Streak Mesoderm (PSM) markers (Brachyury, MIXL1, FOXF1) were upregulated and markers of pluripotency (Nanog, Oct4, Sox2) were downregulated, both compared to the control group. PSM cells nucleofected with Br (PSM-Br) cultured in TF hydrogels retained the NC phenotype consistently for up to 8 weeks, as seen in the gene expression analysis. PSM-Br cells were co-cultured with bone marrow (BM)-derived mesenchymal stem cells (MSCs) which, with time, expressed the NC markers in higher levels, however the levels of expression in BM-MSCs alone did not change. Higher expression of NC and NP marker genes in human BM-MSCs was found to be induced by iNC-condition media (iNC-CM) than porcine NC-CM. The annular puncture induced IVD degeneration as early as 2 weeks after the procedure. The injected iNCs were detected in the degenerated discs after 8 weeks in vivo. The iNC-treated discs were found protected from degeneration. This was evident in histological analysis and changes in the pH levels, indicative of degeneration state of the discs, observed using qCEST MRI. Immunofluorescence stains show that their phenotype was consistent with the in vitro study, namely they still expressed the notochordal markers Keratin 18, Keratin 19, Noto and Brachyury. Conclusion: In the present study, we report a stepwise differentiation method to generate notochordal cells from human iPSCs. These cells not only demonstrate a sustainable notochordal cell phenotype in vitro and in vivo, but also show the functionality of notochordal cells and have protective effect in case of induced disc degeneration and prevent the change in the pH level of the injected IVDs. The mechanism of this effect could be suggested via the paracrine effect on resident cells, as it was shown in the in vitro studies with MSCs.

Background: There is a high incidence of posttraumatic osteoarthritis (PTOA) after anterior cruciate ligament (ACL) injury, and these injuries represent an enormous health care economic burden. In an effort to address this unmet clinical need, there has been increasing interest in cell-based therapies. Purpose: To establish a translational large animal model of PTOA and demonstrate the feasibility of intra-articular human cell–based interventions. Study Design: Descriptive laboratory study. Methods: Nine Yucatan mini-pigs underwent unilateral ACL transection and were monitored for up to 12 weeks after injury. Interleukin 1 beta (IL-1β) levels and collagen breakdown were evaluated longitudinally using enzyme-linked immunosorbent assays of synovial fluid, serum, and urine. Animals were euthanized at 4 weeks (n = 3) or 12 weeks (n = 3) after injury, and injured and uninjured limbs underwent magnetic resonance imaging (MRI) and histologic analysis. At 2 days after ACL injury, an additional 3 animals received an intra-articular injection of 107 human bone marrow–derived mesenchymal stem cells (hBM-MSCs) combined with a fibrin carrier. These cells were labeled with the luciferase reporter gene (hBM-MSCs-Luc) as well as fluorescent markers and intracellular iron nanoparticles. These animals were euthanized on day 0 (n = 1) or day 14 (n = 2) after injection. hBM-MSC-Luc viability and localization were assessed using ex vivo bioluminescence imaging, fluorescence imaging, and MRI. Results: PTOA was detected as early as 4 weeks after injury. At 12 weeks after injury, osteoarthritis could be detected grossly as well as on histologic analysis. Synovial fluid analysis showed elevation of IL-1β shortly after ACL injury, with subsequent resolution by 2 weeks after injury. Collagen type II protein fragments were elevated in the synovial fluid and serum after injury. hBM-MSCs-Luc were detected immediately after injection and at 2 weeks after injection using fluorescence imaging, MRI, and bioluminescence imaging. Conclusion: This study demonstrates the feasibility of reproducing the chondral changes, intra-articular cytokine alterations, and body fluid biomarker findings consistent with PTOA after ACL injury in a large animal model. Furthermore, we have demonstrated the ability of hBM-MSCs to survive and express transgene within the knee joint of porcine hosts without immunosuppression for at least 2 weeks. Clinical Relevance: This model holds great potential to significantly contribute to investigations focused on the development of cell-based therapies for human ACL injury–associated PTOA in the future (see Appendix Figure A1 , available online).

Introduction Intervertebral disc (IVD) degeneration is often associated with low back pain and radiating leg pain. The purpose of this study is to develop a reproducible and standardized preclinical model of painful lumbar IVD degeneration by evaluation of structural and behavioral changes in response to IVD injury with increasing needle sizes. This model can be used to develop new therapies for IVD degeneration. Methods Forty‐five female Sprague Dawley rats underwent anterior lumbar disc needle puncture at levels L4‐5 and L5‐6 under fluoroscopic guidance. Animals were randomly assigned to four different experimental groups: needle sizes of 18 Gauge (G), 21G, 23G, and sham control. To monitor the progression of IVD degeneration and pain, the following methods were employed: μMRI, qRT‐PCR, histology, and biobehavioral analysis. Results T1‐ and T2‐weighted μMRI analysis showed a correlation between the degree of IVD degeneration and needle diameter, with the most severe degeneration in the 18G group. mRNA expression of markers for IVD degeneration markers were dysregulated in the 18G and 21G groups, while pro‐nociceptive markers were increased in the 18G group only. Hematoxylin and Eosin (H&E) and Alcian Blue/Picrosirius Red staining confirmed the most pronounced IVD degeneration in the 18G group. Randall‐Selitto and von Frey tests showed increased hindpaw sensitivity in the 18G group. Conclusion Our findings demonstrate that anterior disc injury with an 18G needle creates severe IVD degeneration and mechanical hypersensitivity, while the 21G needle results in moderate degeneration with no increased pain sensitivity. Therefore, needle sizes should be selected depending on the desired phenotype for the pre‐clinical model., Study Design Overview This study establishes and validates a rat model for lumbar IVD degeneration that is reflected in painful behavior. Based on our findings we conclude that annular disc injury with a 18G needle creates severe, painful IVD degeneration, while 21G needle injury results in moderate degeneration.

Purpose To identify, characterize, and compare the resident progenitor cell populations within the red−red, red−white, and white−white (WW) zones of freshly harvested human cadaver menisci and to characterize the vascularity of human menisci using immunofluorescence and 3-dimensional (3D) imaging. Methods Fresh adult human menisci were harvested from healthy donors. Menisci were enzymatically digested, mononuclear cells isolated, and characterized using flow cytometry with antibodies against mesenchymal stem cell surface markers (CD105, CD90, CD44, and CD29). Cells were expanded in culture, characterized, and compared with bone marrow−derived mesenchymal stem cells. Trilineage differentiation potential of cultured cells was determined. Vasculature of menisci was mapped in 3D using a modified uDisco clearing and immunofluorescence against vascular markers CD31, lectin, and alpha smooth muscle actin. Results There were no significant differences in the clonogenicity of isolated cells between the 3 zones. Flow cytometry showed presence of CD44+CD105+CD29+CD90+ cells in all 3 zones with high prevalence in the WW zone. Progenitors from all zones were found to be potent to differentiate to mesenchymal lineages. Larger vessels in the red−red zone of meniscus were observed spanning toward red−white, sprouting to smaller arterioles and venules. CD31+ cells were identified in all zones using the 3D imaging and co-localization of additional markers of vasculature (lectin and alpha smooth muscle actin) was observed. Conclusions The presence of resident mesenchymal progenitors was evident in all 3 meniscal zones of healthy adult donors without injury. In addition, our results demonstrate the presence of vascularization in the WW zone. Clinical Relevance The existence of progenitors and presence of microvasculature in the WW zone of the meniscus suggests the potential for repair and biologic augmentation strategies in that zone of the meniscus in young healthy adults. Further research is necessary to fully define the functionality of the meniscal blood supply and its implications for repair.

Intervertebral disc (IVD) degeneration is a leading cause of chronic low back pain that affects millions of people every year. Yet identification of the specific IVD causing this pain is based on qualitative visual interpretation rather than objective findings. One possible approach to diagnosing pain-associated IVD could be to identify acidic IVDs, as decreased pH within an IVD has been postulated to mediate discogenic pain. We hypothesized that quantitative chemical exchange saturation transfer (qCEST) MRI could detect pH changes in IVDs, and thence be used to diagnose pathologically painful IVDs objectively and noninvasively. To test this hypothesis, a surgical model of IVD degeneration in Yucatan minipigs was used. Direct measurement of pH inside the degenerated IVDs revealed a significant drop in pH after degeneration, which correlated with a significant increase in the qCEST signal. Gene analysis of harvested degenerated IVDs revealed significant upregulation of pain-, nerve- and inflammatory-related markers after IVD degeneration. A strong positive correlation was observed between the expression of pain markers and the increase in the qCEST signal. Collectively, these findings suggest that this approach might be used to identify which IVD is causing low back pain, thereby providing valuable guidance for pain and surgical management.

Background: Although tendon injuries and repairs are common, treatment of these injuries has limitations. The application of mesenchymal progenitor cells (MPCs) is increasingly used to optimize the biological process of tendon repair healing. However, clinically relevant technologies that effectively assess the localization of exogenous MPCs in vivo are lacking. Hypothesis: Exogenous MPCs labeled with superparamagnetic iron oxide (SPIO) particles would allow monitoring of the localization and retention of cells within the site of implantation via magnetic resonance imaging (MRI) without negatively affecting cell survival or differentiation. Study Design: Descriptive laboratory study. Methods: Genetically modified C3H10T1/2 MPCs engineered to express luciferase (Luc+) reporter gene were implanted into surgically created Achilles tendon defects of 10 athymic nude rats (Hsd:RH-Foxn1rnu). Of these animals, 5 animals received Luc+ C3H10T1/2 MPCs colabeled with SPIO nanoparticles (+SPIO). These 2 groups of animals then underwent optical imaging with quantification of bioluminescence and MRI at 7, 14, and 28 days after surgery. Statistical analysis was conducted by use of 2-way analysis of variance. At 28 days after surgery, animals were euthanized and the treated limbs underwent histologic analysis. Results: Optical imaging demonstrated that the implanted cells not only survived but also proliferated in vivo, and these cells remained viable for at least 4 weeks after implantation. In addition, SPIO labeling did not appear to affect MPC survival or proliferation, as assessed by quantitative bioluminescence imaging ( P > .05, n = 5). MRI demonstrated that SPIO labeling was an effective method to monitor cell localization, retention, and viability for at least 4 weeks after implantation. Histologic and immunofluorescence analyses of the repaired tendon defect sites demonstrated tenocyte-like labeled cells, suggesting that cell differentiation was not affected by labeling the cells with the SPIO nanoparticles. Conclusion: MRI of exogenous MPCs labeled with SPIO particles allows for effective in vivo assessments of cell localization and retention in the setting of tendon regeneration for at least 4 weeks after implantation. This SPIO labeling does not appear to impair cell survival, transgene expression, or differentiation. Clinical Relevance: SPIO labeling of MPCs appears to be safe for in vivo assessments of MPCs in tendon regeneration therapies and may be used for future clinical investigations of musculoskeletal regenerative medicine.

Purpose Previous studies have associated low pH in intervertebral discs (IVDs) with discogenic back pain. The purpose of this study was to determine whether quantitative CEST (qCEST) MRI can be used to detect pH changes in IVDs in vivo. Methods The exchange rate ksw between glycosaminoglycan (GAG) protons and water protons was determined from qCEST analysis. Its dependence on pH value was investigated in GAG phantoms with varying pH and concentrations. The relationship between ksw and pH was studied further in vivo in a porcine model on a 3T MR scanner and validated using a pH meter. Sodium lactate was injected into the IVDs to induce various pH values within the discs ranging from 5 to 7. Results Phantom and animal results revealed that ksw measured using qCEST MRI is highly correlated with pH level. In the animal studies, the relationship can be described as ksw =9.2 × 106 × 10−pH + 196.9, R2 = 0.7883. Conclusion The exchange rate between GAG and water protons determined from qCEST MRI is closely correlated with pH value. This technique has the potential to noninvasively measure pH in the IVDs of patients with discogenic pain. Magn Reson Med, 2016. © 2016 International Society for Magnetic Resonance in Medicine

Using short-term exposure of embryoid bodies to transforming growth factor-β, the authors directed induced pluripotent stem cells (iPSCs) toward mesenchymal stem cell (MSC) differentiation. Two types of iPSC-derived MSCs were identified: early (aiMSCs) and late (tiMSCs) outgrowing cells. Both types differentiated in vitro in response to osteogenic or adipogenic supplements; aiMSCs demonstrated higher osteogenic potential than tiMSCs. Upon orthotopic injection into radial defects, both types regenerated bone and contributed to defect repair., Mesenchymal stem cells (MSCs) are currently the most established cells for skeletal tissue engineering and regeneration; however, their availability and capability of self-renewal are limited. Recent discoveries of somatic cell reprogramming may be used to overcome these challenges. We hypothesized that induced pluripotent stem cells (iPSCs) that were differentiated into MSCs could be used for bone regeneration. Short-term exposure of embryoid bodies to transforming growth factor-β was used to direct iPSCs toward MSC differentiation. During this process, two types of iPSC-derived MSCs (iMSCs) were identified: early (aiMSCs) and late (tiMSCs) outgrowing cells. The transition of iPSCs toward MSCs was documented using MSC marker flow cytometry. Both types of iMSCs differentiated in vitro in response to osteogenic or adipogenic supplements. The results of quantitative assays showed that both cell types retained their multidifferentiation potential, although aiMSCs demonstrated higher osteogenic potential than tiMSCs and bone marrow-derived MSCs (BM-MSCs). Ectopic injections of BMP6-overexpressing tiMSCs produced no or limited bone formation, whereas similar injections of BMP6-overexpressing aiMSCs resulted in substantial bone formation. Upon orthotopic injection into radial defects, all three cell types regenerated bone and contributed to defect repair. In conclusion, MSCs can be derived from iPSCs and exhibit self-renewal without tumorigenic ability. Compared with BM-MSCs, aiMSCs acquire more of a stem cell phenotype, whereas tiMSCs acquire more of a differentiated osteoblast phenotype, which aids bone regeneration but does not allow the cells to induce ectopic bone formation (even when triggered by bone morphogenetic proteins), unless in an orthotopic site of bone fracture. Significance Mesenchymal stem cells (MSCs) are currently the most established cells for skeletal tissue engineering and regeneration of various skeletal conditions; however, availability of autologous MSCs is very limited. This study demonstrates a new method to differentiate human fibroblast-derived induced pluripotent stem cells (iPSCs) to cells with MSC properties, which we comprehensively characterized including differentiation potential and transcriptomic analysis. We showed that these iPS-derived MSCs are able to regenerate nonunion bone defects in mice more efficiently than bone marrow-derived human MSCs when overexpressing BMP6 using a nonviral transfection method.

BACKGROUND CONTEXT Intervertebral disc (IVD) degeneration is often associated with low back pain and radiating leg pain. Disc puncture injury rat models are used to study IVD degeneration, which depend highly on the needle diameter and mode of insertion. PURPOSE The purpose of this study is to develop a reproducible and standardized pre-clinical model of painful lumbar IVD degeneration by evaluation of structural and behavioral changes in response to IVD injury with increasing needle sizes. This model can be used to develop new therapies for IVD degeneration. STUDY DESIGN/SETTING In-vivo animal study. PATIENT SAMPLE Not applicable. OUTCOME MEASURES µMRI, qRT-PCR, histology and biobehavioral analysis. METHODS Forty-five Sprague Dawley rats underwent anterior lumbar disc needle puncture at levels L4-5 and L5-6 under fluoroscopic guidance. Animals were randomly assigned to 4 different experimental groups: needle sizes of 18 gauge (G), 21G, 23G and sham control. To monitor the progression of IVD degeneration and pain, the following methods were employed: µMRI, qRT-PCR, histology and biobehavioral analysis. RESULTS T1- and T2-weighted µMRI analysis showed a correlation between the degree of IVD degeneration and needle diameter, with the most severe degeneration in the 18G group. mRNA expression of markers for IVD degeneration markers were dysregulated in the 18G and 21G groups, while pro-nociceptive markers were increased in the 18G group only. H&E and Alcian Blue/Picrosirius Red staining confirmed the most pronounced IVD degeneration in the 18G group. Randall-Selitto and von Frey tests showed increased hind paw sensitivity in the 18G group. CONCLUSIONS Our findings demonstrate that anterior disc injury with an 18G needle creates severe IVD degeneration and mechanical hypersensitivity, while the 21G needle results in moderate degeneration with no behavioral changes. Needle sizes should therefore be selected depending on the desired phenotype for the pre-clinical model. FDA DEVICE/DRUG STATUS This abstract does not discuss or include any applicable devices or drugs.

Ligament injuries occur frequently, substantially hindering routine daily activities and sports participation in patients. Surgical reconstruction using autogenous or allogeneic tissues is the gold standard treatment for ligament injuries. Although surgeons routinely perform ligament reconstructions, the integrity of these reconstructions largely depends on adequate biological healing of the interface between the ligament graft and the bone. We hypothesized that localized ultrasound-mediated, microbubble-enhanced therapeutic gene delivery to endogenous stem cells would lead to significantly improved ligament graft integration. To test this hypothesis, an anterior cruciate ligament reconstruction procedure was performed in Yucatan mini-pigs. A collagen scaffold was implanted in the reconstruction sites to facilitate recruitment of endogenous mesenchymal stem cells. Ultrasound-mediated reporter gene delivery successfully transfected 40% of cells recruited to the reconstruction sites. When BMP-6 encoding DNA was delivered, BMP-6 expression in the reconstruction sites was significantly enhanced. Micro-computed tomography and biomechanical analyses showed that ultrasound-mediated BMP-6 gene delivery led to significantly enhanced osteointegration in all animals 8 weeks after surgery. Collectively, these findings demonstrate that ultrasound-mediated gene delivery to endogenous mesenchymal progenitor cells can effectively improve ligament reconstruction in large animals, thereby addressing a major unmet orthopedic need and offering new possibilities for translation to the clinical setting., Molecular Therapy, 26 (7), ISSN:1525-0016, ISSN:1525-0024

Disorders of the temporomandibular joint (TMJ) complex affect 6-12% of the population; the joint's disc is usually involved. Tissue engineering and regenerative medicine may constitute a promising therapeutic approach, with resident stromal progenitor cells a key factor in the process. We hypothesized that the TMJ disc (TMJD) contains multipotent stromal progenitors that may play an important role in regeneration of the disc. TMJD cells were cultured and evaluated for growth kinetics and colony-forming units (CFUs). Single cell-derived clones were isolated and induced to differentiate toward the osteogenic, adipogenic and chondrogenic lineages by culturing in various induction media. Flow cytometry was used to identify multipotent stromal cell surface markers in additional cell samples, and reverse transcription-polymerase chain reaction (RT-PCR) was used to determine gene expression patterns within isolated cells. High numbers of CFUs were observed, indicating cell self-renewal. Biochemical assays showed significantly higher alkaline phosphatase (ALP) activity, lipid droplet concentration and glycosaminoglycan levels in cells cultured in osteogenic, adipogenic and chondrogenic induction medium, respectively. Approximately 1% of the total cell population demonstrated the capability to differentiate into all three mesenchymal lineages. Chondrogenic gene levels within TMJD-derived cells were significantly reduced in passaged culture. Our results support the hypothesis that multipotent stromal progenitor cells populate the TMJD and possess proliferation and differentiation capabilities. These cells may contribute to the regeneration potential of dysfunctional tissue and become the primary component in future attempts at tissue engineering or regeneration of this complex. Copyright © 2015 John Wiley & Sons, Ltd.

Osteoporosis-related vertebral compression fractures (OVCFs) are a common and clinically unmet need with increasing prevalence as the world population ages. Animal OVCF models are essential to the preclinical development of translational tissue engineering strategies. While a number of models currently exist, this protocol describes an optimized method for inducing multiple highly reproducible vertebral defects in a single nude rat. A novel longitudinal semiautomated microcomputed tomography (µCT)-based quantitative structural analysis of the vertebral defects is also detailed. Briefly, rats were imaged at multiple time points post-op. The day 1 scan was reoriented to a standard position, and a standard volume of interest was defined. Subsequent µCT scans of each rat were automatically registered to the day 1 scan so the same volume of interest was then analyzed to assess for new bone formation. This versatile approach can be adapted to a variety of other models where longitudinal imaging-based analysis could benefit from precise 3D semiautomated alignment. Taken together, this protocol describes a readily quantifiable and easily reproducible system for osteoporosis and bone research. The suggested protocol takes 4 months to induce osteoporosis in nude ovariectomized rats and between 2.7 and 4 h to generate, image, and analyze two vertebral defects, depending on tissue size and equipment.

The oscillation of microbubbles has long been hypothesized to provide the opportunity to enhance gene delivery as a result of changes in membrane permeability; however, translationally-relevant therapeutic protocols have not yet been realized. We sought to develop and validate a protocol to transfect endogenous mesenchymal stem cells (MSCs) via the local injection of plasmids and microbubbles and the application of ultrasound. We apply this therapy in a pre-clinical model to solve an important clinical problem — that of healing segmental bone defects. More than two million bone-grafting procedures are performed each year using autografts or allografts and these standard of care therapies have substantial disadvantages.

More than 2 million bone-grafting procedures are performed each year using autografts or allografts. However, both options carry disadvantages, and there remains a clear medical need for the development of new therapies for massive bone loss and fracture nonunions. We hypothesized that localized ultrasound-mediated, microbubble-enhanced therapeutic gene delivery to endogenous stem cells would induce efficient bone regeneration and fracture repair. To test this hypothesis, we surgically created a critical-sized bone fracture in the tibiae of Yucatán mini-pigs, a clinically relevant large animal model. A collagen scaffold was implanted in the fracture to facilitate recruitment of endogenous mesenchymal stem/progenitor cells (MSCs) into the fracture site. Two weeks later, transcutaneous ultrasound-mediated reporter gene delivery successfully transfected 40% of cells at the fracture site, and flow cytometry showed that 80% of the transfected cells expressed MSC markers. Human bone morphogenetic protein-6 (BMP-6) plasmid DNA was delivered using ultrasound in the same animal model, leading to transient expression and secretion of BMP-6 localized to the fracture area. Micro–computed tomography and biomechanical analyses showed that ultrasound-mediated BMP-6 gene delivery led to complete radiographic and functional fracture healing in all animals 6 weeks after treatment, whereas nonunion was evident in control animals. Collectively, these findings demonstrate that ultrasound-mediated gene delivery to endogenous mesenchy-mal progenitor cells can effectively treat nonhealing bone fractures in large animals, thereby addressing a major orthopedic unmet need and offering new possibilities for clinical translation.

Background A devastating condition that leads to trauma-related morbidity, multiple rib fractures, remain a serious unmet clinical need. Systemic administration of mesenchymal stem cells (MSCs) has been shown to regenerate various tissues. We hypothesized that parathyroid hormone (PTH) therapy would enhance MSC homing and differentiation, ultimately leading to bone formation that would bridge rib fractures. Methods The combination of human MSCs (hMSCs) and a clinically relevant PTH dose was studied using immunosuppressed rats. Segmental defects were created in animals’ fifth and sixth ribs. The rats were divided into four groups: a negative control group, in which animals received vehicle alone; the PTH-only group, in which animals received daily subcutaneous injections of 4 μg/kg teriparatide, a pharmaceutical derivative of PTH; the hMSC-only group, in which each animal received five injections of 2 × 106 hMSCs; and the hMSC + PTH group, in which animals received both treatments. Longitudinal in vivo monitoring of bone formation was performed biweekly using micro-computed tomography (μCT), followed by histological analysis. Results Fluorescently-dyed hMSCs were counted using confocal microscopy imaging of histological samples harvested 8 weeks after surgery. PTH significantly augmented the number of hMSCs that homed to the fracture site. Immunofluorescence of osteogenic markers, osteocalcin and bone sialoprotein, showed that PTH induced cell differentiation in both exogenously administered cells and resident cells. μCT scans revealed a significant increase in bone volume only in the hMSC + PTH group, beginning by the 4th week after surgery. Eight weeks after surgery, 35% of ribs in the hMSC + PTH group had complete bone bridging, whereas there was complete bridging in only 6.25% of ribs (one rib) in the PTH-only group and in none of the ribs in the other groups. Based on the μCT scans, biomechanical analysis using the micro-finite element method demonstrated that the healed ribs were stiffer than intact ribs in torsion, compression, and bending simulations, as expected when examining bone callus composed of woven bone. Conclusions Administration of both hMSCs and PTH worked synergistically in rib fracture healing, suggesting this approach may pave the way to treat multiple rib fractures as well as additional fractures in various anatomical sites. Electronic supplementary material The online version of this article (doi:10.1186/s13287-017-0502-9) contains supplementary material, which is available to authorized users.

When fractured, bone can spontaneously heal to a certain extent. In critical-size defects, in which bone loss is severe, a bone graft is required. Faced with the shortcomings of grafts currently in use—autografts, allografts, and mineral-based bone substitutes—efforts are being made to establish new methods of bone regeneration. One promising approach involves the use of exogenous gene-modified mesenchymal stem cells (MSCs), which can rapidly repair large bone defects in animal models. Unfortunately, ex vivo culture of MSCs may add certain complexity to the advancement of this cell therapy to the clinic. Previously, we demonstrated efficient bone regeneration following direct gene delivery to endogenous MSCs that had been attracted to a fracture site. In that study, electroporation was used, but it is an invasive method of gene transfection that may result in tissue damage. Unlike electroporation, sonoporation—the use of ultrasound for gene delivery—is noninvasive, considered safer, and relevant to the clinical setting. In this study, we evaluated the feasibility of ultrasound-based gene delivery to resident MSCs that had been recruited to a fracture site in different animal models. Our results show transient (up to 21 days) expression of a reporter gene in radial, vertebral, and tibial bone defects in mice, rats, and a minipig. These results could prove beneficial for the next phase of experiments, in which osteogenic genes such as the bone morphogentic proteins (BMPs) could be utilized to achieve bone regeneration in similar challenging conditions of bone tissue loss. Copyright © 2014 John Wiley & Sons, Ltd.

Purpose Low pH is associated with intervertebral disc (IVD)-generated low back pain (LBP). The purpose of this work was to develop an in vivo pH level-dependent magnetic resonance imaging (MRI) method for detecting discogenic LBP, without using exogenous contrast agents.

The reduced field-of-view (rFOV) turbo-spin-echo (TSE) technique, which effectively suppresses bowel movement artifacts, is developed for the purpose of chemical exchange saturation transfer (CEST) imaging of the intervertebral disc (IVD) in vivo. Attempts to quantify IVD CEST signals in a clinical setting require high reliability and accuracy, which is often compromised in the conventionally used technique. The proposed rFOV TSE CEST method demonstrated significantly superior reproducibility when compared with the conventional technique on healthy volunteers, implying it is a more reliable measurement. Phantom study revealed a linear relation between CEST signal and glycosaminoglycan (GAG) concentration. The feasibility of detecting IVD degeneration was demonstrated on a healthy volunteer, indicating that the proposed method is a promising tool to quantify disc degeneration.

Background: Brown adipose tissue plays a pivotal role in mammal metabolism and thermogenesis. It has a great therapeutic potential in several metabolic disorders such as obesity and diabetes. Mesenchymal stem cells (MSCs) are suitable candidates for brown adipose tissue formation de novo. Pparγ2 and C/ebpα are nucleic receptors known to mediate adipogenic differentiation. We hypothesized that overexpression of the Pparγ2 and C/ebpα genes in MSCs would lead to the formation of adipose tissue. Materials & methods: MSCs bearing the Luc reporter gene were transfected to overexpress Pparγ2 and C/ebpα. Differentiation of nucleofected cells was evaluated in vitro and in vivo following ectopic implantation of the cells in C3H/HeN mice. Results: After implantation, the engineered cells survived for 5 weeks and brown adipose-like tissue was observed in histological samples. Immunostaining and bioluminescent imaging showed new adipocytes expressing Luc and the brown adipose tissue marker, UCP1, in vitro and in vivo. Conclusion: We show that gene delivery of transcription factors into MSCs generates brown adipose tissue in vitro and in vivo.

Osteoporosis affects more than 200 million people worldwide leading to more than 2 million fractures in the United States alone. Unfortunately, surgical treatment is limited in patients with low bone mass. Parathyroid hormone (PTH) was shown to induce fracture repair in animals by activating mesenchymal stem cells (MSCs). However, it would be less effective in patients with fewer and/or dysfunctional MSCs due to aging and comorbidities. To address this, we evaluated the efficacy of combination i.v. MSC and PTH therapy versus monotherapy and untreated controls, in a rat model of osteoporotic vertebral bone defects. The results demonstrated that combination therapy significantly increased new bone formation versus monotherapies and no treatment by 2 weeks (P < 0.05). Mechanistically, we found that PTH significantly enhanced MSC migration to the lumbar region, where the MSCs differentiated into bone-forming cells. Finally, we used allogeneic porcine MSCs and observed similar findings in a clinically relevant minipig model of vertebral defects. Collectively, these results demonstrate that in addition to its anabolic effects, PTH functions as an adjuvant to i.v. MSC therapy by enhancing migration to heal bone loss. This systemic approach could be attractive for various fragility fractures, especially using allogeneic cells that do not require invasive tissue harvest.

A major parameter determining the success of a bone-grafting procedure is vascularization of the area surrounding the graft. We hypothesized that implantation of a bone autograft would induce greater bone regeneration by abundant blood vessel formation. To investigate the effect of the graft on neovascularization at the defect site, we developed a micro-computed tomography (µCT) approach to characterize newly forming blood vessels, which involves systemic perfusion of the animal with a polymerizing contrast agent. This method enables detailed vascular analysis of an organ in its entirety. Additionally, blood perfusion was assessed using fluorescence imaging (FLI) of a blood-borne fluorescent agent. Bone formation was quantified by FLI using a hydroxyapatite-targeted probe and µCT analysis. Stem cell recruitment was monitored by bioluminescence imaging (BLI) of transgenic mice that express luciferase under the control of the osteocalcin promoter. Here we describe and demonstrate preparation of the allograft, calvarial defect surgery, µCT scanning protocols for the neovascularization study and bone formation analysis (including the in vivo perfusion of contrast agent), and the protocol for data analysis. The 3D high-resolution analysis of vasculature demonstrated significantly greater angiogenesis in animals with implanted autografts, especially with respect to arteriole formation. Accordingly, blood perfusion was significantly higher in the autograft group by the 7(th) day after surgery. We observed superior bone mineralization and measured greater bone formation in animals that received autografts. Autograft implantation induced resident stem cell recruitment to the graft-host bone suture, where the cells differentiated into bone-forming cells between the 7(th) and 10(th) postoperative day. This finding means that enhanced bone formation may be attributed to the augmented vascular feeding that characterizes autograft implantation. The methods depicted may serve as an optimal tool to study bone regeneration in terms of tightly bounded bone formation and neovascularization.

Nonunion fractures present a challenge to orthopedics with no optimal solution. In-vivo DNA electroporation is a gene-delivery technique that can potentially accelerate regenerative processes. We hypothesized that in vivo electroporation of an osteogenic gene in a nonunion radius bone defect site would induce fracture repair. Nonunion fracture was created in the radii of C3H/HeN mice, into which a collagen sponge was placed. To allow for recruitment of host progenitor cells (HPCs) into the implanted sponge, the mice were housed for 10 days before electroporation. Mice were electroporated with either bone morphogenetic protein 9 (BMP-9) plasmid, Luciferase plasmid or injected with BMP-9 plasmid but not electroporated. In vivo bioluminescent imaging indicated that gene expression was localized to the defect site. Microcomputed tomography (µCT) and histological analysis of murine radii electroporated with BMP-9 demonstrated bone formation bridging the bone gap, whereas in the control groups the defect remained unbridged. Population of the implanted collagen sponge by HPCs transfected with the injected plasmid following electroporation was noted. Our data indicate that regeneration of nonunion bone defect can be attained by performing in vivo electroporation with an osteogenic gene combined with recruitment of HPCs. This gene therapy approach may pave the way for regeneration of other skeletal tissues.

Microcomputed tomography (microCT) analysis is a powerful tool for the evaluation of bone tissue because it provides access to the 3D microarchitecture of the bone. It is invaluable for regenerative medicine as it provides the researcher with the opportunity to explore the skeletal system both in vivo and ex vivo. The quantitative assessment of macrostructural characteristics and microstructural features may improve our ability to estimate the quality of newly formed bone. We have developed a unique procedure for analyzing data from microCT scans to evaluate bone structure and repair. This protocol describes the procedures for microCT analysis of three main types of mouse bone regeneration models (ectopic administration of bone-forming mesenchymal stem cells, and administration of cells after both long bone defects and cranial segmental bone defects) that can be easily adapted for a variety of other models. Precise protocols are crucial because the system is extremely user sensitive and results can be easily biased if standardized methods are not applied. The suggested protocol takes 1.5-3.5 h per sample, depending on bone tissue sample size, the type of equipment used, variables of the scanning protocol and the operator's experience.

Most spine fusion procedures involve the use of prosthetic fixation devices combined with autologous bone grafts rather than biological treatment. We had shown that spine fusion could be achieved by injection of bone morphogenetic protein-2 (BMP-2)-expressing mesenchymal stem cells (MSCs) into the paraspinal muscle. In this study, we hypothesized that posterior spinal fusion achieved using genetically modified MSCs would be mechanically comparable to that realized using a mechanical fixation. BMP-2-expressing MSCs were injected bilaterally into paravertebral muscles of the mouse lumbar spine. In one control group BMP-2 expression was inhibited. Microcomputed tomography and histological analyses were used to evaluate bone formation. For comparison, a group of mouse spines were bilaterally fused with stainless steel pins. The harvested spines were later tested using a custom four-point bending apparatus and structural bending stiffness was estimated. To assess the degree to which MSC vertebral fusion was targeted and to quantify the effects of fusion on adjacent spinal segments, images of the loaded spine curvature were analyzed to extract rigidity of the individual spinal segments. Bone bridging of the targeted vertebrae was observed in the BMP-2-expressing MSC group, whereas no bone formation was noted in any control group. The biomechanical tests showed that MSC-mediated spinal fusion was as effective as stainless steel pin-based fusion and significantly more rigid than the control groups. Local analysis showed that the distribution of stiffness in the MSC-based fusion group was similar to that in the steel pin fusion group, with the majority of spinal stiffness contributed by the targeted fusion at L3-L5. Our findings demonstrate that MSC-induced spinal fusion can convey biomechanical rigidity to a targeted segment that is comparable to that achieved using an instrumental fixation.

More than 1800 gene therapy clinical trials worldwide have targeted a wide range of conditions including cancer, cardiovascular diseases, and monogenic diseases. Biological (i.e. viral), chemical, and physical approaches have been developed to deliver nucleic acids into cells. Although viral vectors offer the greatest efficiency, they also raise major safety concerns including carcinogenesis and immunogenicity. The goal of microbubble-mediated sonoporation is to enhance the uptake of drugs and nucleic acids. Insonation of microbubbles is thought to facilitate two mechanisms for enhanced uptake: first, deflection of the cell membrane inducing endocytotic uptake, and second, microbubble jetting inducing the formation of pores in the cell membrane. We hypothesized that ultrasound could be used to guide local microbubble-enhanced sonoporation of a reporter gene encoding DNA. With the aim of optimizing delivery efficiency, we used nonlinear ultrasound and bioluminescence imaging modes to optimize the acoustic pressure, microbubble concentration, treatment duration, DNA dosage, and number of treatments required for in vivo Luciferase gene expression in a mouse thigh muscle model. We found that mice injected with 50 μg luciferase plasmid DNA and 5 x 105 microbubbles followed by ultrasound treatment at 1.4 MHz, 200 kPa, 100-cycle pulse length, and 540-Hz pulse repetition frequency (PRF) for 2 min exhibited superior transgene expression compared to all other treatment groups. The bioluminescent signal measured for these mice on Day 4 post-treatment was 100-fold higher (p < 0.0001, n = 5 or 6) than the signals for controls treated with DNA injection alone, DNA and microbubble injection, or DNA injection and ultrasound treatment. Our results indicate that these conditions result in efficient gene delivery and prolonged gene expression (up to 21 days) with no evidence of tissue damage or off-target delivery. We believe that these promising results bear great promise for the development of microbubble-enhanced sonoporation–induced gene therapies.

Objectives: The ultimate healing of a ligament graft to bone is a lengthy process that results in reparative tissue that is distinctly different from a native ligament insertion. This known structural aberration results in inferior biomechanical properties and can contribute significantly to recurrent injury as well as compromised function. Thus, technology allowing for accelerated biologic incorporation as well as the recapitulation of a native insertion would be of great benefit to patients. We hypothesized that the treatment of anterior cruciate ligament (ACL) reconstruction bone tunnels with bone morphogenetic protein (BMP) DNA combined with a pulse of ultrasound (US) energy would lead to activation of endogenous stem cells and result in enhanced graft incorporation. Methods: Soft tissue allograft ACL reconstruction was performed in nine mini-pigs using cortical screw post fixation. A collagen scaffold was added to the terminus of each tendon graft limb contained within the corresponding bone tunnel. On post-operative day 14, a lipid microsphere solution containing DNA encoding either green fluorescence protein (GFP, n=3 pigs) or BMP-6 (n=6 pigs) was delivered into the bone tunnels percutaneously under fluoroscopic guidance. The bone tunnels in the US treated group were then immediately treated with a transcutaneous US energy pulse at the site of the bone tunnels, whereas the control group did not receive US delivery. At five days post-treatment the cells occupying the bone tunnels of the GFP treated pigs (n=3) were harvested and subjected to fluorescence-activated cell sorting (FACS) analysis to assess for expression of GFP as well as known mesenchymal stem cell (MSC) markers (CD29, CD44, CD90). Six weeks post-treatment the BMP/US treated pigs (n=3) and control pigs (n=3) were sacrificed and their knee joints were scanned using microCT. Biomechanical testing of anterior-posterior (AP) knee laxity was performed via application of sinusoidal AP-directed shear loads for 12 cycles. Load to failure was assessed by removal of the joint capsule, menisci, collateral ligaments, and the PCL leaving only the ACL graft intact. The tibia and femur were then mounted on a universal material testing machine with a 5kN load cell and distracted until failure of the ACL graft or graft-bone interface. Results: FACS analysis done 5 days after GFP gene delivery showed that 40-50% of the cells occupying the US treated bone tunnels expressed GFP and this was significantly more than the cells in untreated bone tunnels (p< 0.05). In addition, 12-22% of the cells in the bone tunnels expressed MSC markers. Quantification of bone volume in ACL reconstruction tunnels showed significantly higher values in BMP-6/US treated animals (fig 1A,B,C; *pConclusion: Endogenous stem cell gene expression can be altered in a large animal model via novel minimally invasive techniques and this can result in enhanced ligament biomechanical properties. Minimally invasive biologic technology such as this would likely be of significant benefit to the treatment of patients undergoing ligament reconstruction procedures and more research regarding these technologies is certainly warranted.

Disorders of the temporomandibular joint (TMJ) complex affect 6-12% of the population; the joint's disc is usually involved. Tissue engineering and regenerative medicine may constitute a promising therapeutic approach, with resident stromal progenitor cells a key factor in the process. We hypothesized that the TMJ disc (TMJD) contains multipotent stromal progenitors that may play an important role in regeneration of the disc. TMJD cells were cultured and evaluated for growth kinetics and colony-forming units (CFUs). Single cell-derived clones were isolated and induced to differentiate toward the osteogenic, adipogenic and chondrogenic lineages by culturing in various induction media. Flow cytometry was used to identify multipotent stromal cell surface markers in additional cell samples, and reverse transcription-polymerase chain reaction (RT-PCR) was used to determine gene expression patterns within isolated cells. High numbers of CFUs were observed, indicating cell self-renewal. Biochemical assays showed significantly higher alkaline phosphatase (ALP) activity, lipid droplet concentration and glycosaminoglycan levels in cells cultured in osteogenic, adipogenic and chondrogenic induction medium, respectively. Approximately 1% of the total cell population demonstrated the capability to differentiate into all three mesenchymal lineages. Chondrogenic gene levels within TMJD-derived cells were significantly reduced in passaged culture. Our results support the hypothesis that multipotent stromal progenitor cells populate the TMJD and possess proliferation and differentiation capabilities. These cells may contribute to the regeneration potential of dysfunctional tissue and become the primary component in future attempts at tissue engineering or regeneration of this complex. Copyright © 2015 John WileySons, Ltd.

While ischemia, hypoxic hypoxia, and carbon monoxide (CO) have received extensive study designed to characterize mechanisms by which they disrupt cochlear function, little data are available concerning cyanide's potential to disrupt auditory function. In this study, disruption of the compound action potential (CAP) and endocochlear potential (EP) by cyanide and CO was compared in rats treated with potassium cyanide (KCN) (7 mg/kg ip), saline, CO (35 ml/kg ip), and air. Acute KCN administration significantly suppressed CAP and EP transiently. The effect was seen initially on EP with CAP impairment occurring a few minutes later. Acute CO injection also suppressed the CAP significantly, but the effect was far smaller, occurred later in time, and lasted longer than the effect of KCN. The effect of CO on EP was equivocal. There was a good correspondence between blood cyanide levels and impairment of cochlear function; carboxyhemoglobin (HbCO) levels were elevated during the period when cochlear function was impaired, but recovery of cochlear function preceded the return of normal oxyhemoglobin. Both KCN and CO had somewhat preferential effects on high-frequency tones. Repeated cyanide administration caused a persistent CAP threshold elevation despite the rapid recovery of EP and CAP observed following acute KCN administration. The data suggest that acute KCN administration has a prominent disruptive effect at the stria vascularis presumably by disrupting the electron transport chain in this metabolically active structure. The principal target for acute CO ototoxicity in the cochlea is probably not the stria vascularis.

Real-time bioluminescence functional imaging holds great promise for regenerative medicine because it improves the researcher's ability to analyze and understand the healing process. Using transgenic mice coupled with gene-modified cells, one can employ this method to monitor host and graft activity in various models of tissue regeneration. We implemented real-time bioluminescence functional imaging to analyze bone formation by following a unique protocol in which the luciferase reporter gene, driven by an osteocalcin promoter, is used to visualize host and graft activity during bone formation. Real-time bioluminescence functional imaging can be used to assess the "host reaction" in transgenic mice models; it can also be used to assess "graft activity" in other animals in which genetically labeled stem cells have been implanted or direct gene delivery has been applied. The suggested imaging protocol requires 25 min per sample. However, special attention must be given to the layout of the experimental design, which determines the specific activity that will be analyzed.

Background context The intervertebral disc (IVD) possesses a minimal capability for self-repair and regeneration. Changes in the differentiation of resident progenitor cells can represent diminished tissue regeneration and a loss of homeostasis. We previously showed that progenitor cells reside in the nucleus pulposus (NP). The effect of the degenerative process on these cells remains unclear. Purpose We sought to explore the effect of IVD degeneration on the abundance of resident progenitor cells in the NP, their differentiation potential, and their ability to give rise to NP-like cells. We hypothesize that disc degeneration affects those properties. Study design Nucleus pulposus cells derived from healthy and degenerated discs were methodically compared for proliferation, differentiation potential, and ability to generate NP-like cells. Methods Intervertebral disc degeneration was induced in 10 skeletally, mature mini pigs using annular injury approach. Degeneration was induced in three target discs, whereas intact adjacent discs served as controls. The disc degeneration was monitored using magnetic resonance imaging for 6 to 8 weeks. After there was a clear evidence of degeneration, we isolated and compared cells from degenerated discs (D-NP cells [NP-derived cells from porcine degenerated discs]) with cells isolated from healthy discs (H-NP cells) obtained from the same animal. Results The comparison showed that D-NP cells had a significantly higher colony-forming unit rate and a higher proliferation rate in vitro. Our data also indicate that although both cell types are able to differentiate into mesenchymal lineages, H-NP cells exhibit significantly greater differentiation toward the chondrogenic lineage and NP-like cells than D-NP cells, displaying greater production of glycosaminoglycans and higher gene expression of aggrecan and collagen IIa. Conclusions Based on these findings, we conclude that IVD degeneration has a meaningful effect on the cells in the NP. D-NP cells clearly go through the regenerative process; however, this process is not powerful enough to facilitate full regeneration of the disc and reverse the degenerative course. These findings facilitate deeper understanding of the IVD degeneration process and trigger further studies that will contribute to development of novel therapies for IVD degeneration.

The reduced field-of-view (rFOV) turbo-spin-echo (TSE) technique, which effectively suppresses bowel movement artifacts, is developed for the purpose of chemical exchange saturation transfer (CEST) imaging of the intervertebral disc (IVD) in vivo. Attempts to quantify IVD CEST signals in a clinical setting require high reliability and accuracy, which is often compromised in the conventionally used technique. The proposed rFOV TSE CEST method demonstrated significantly superior reproducibility when compared with the conventional technique on healthy volunteers, implying it is a more reliable measurement. Phantom study revealed a linear relation between CEST signal and glycosaminoglycan (GAG) concentration. The feasibility of detecting IVD degeneration was demonstrated on a healthy volunteer, indicating that the proposed method is a promising tool to quantify disc degeneration.

Real-time bioluminescence functional imaging holds great promise for regenerative medicine because it improves the researcher's ability to analyze and understand the healing process. Using transgenic mice coupled with gene-modified cells, one can employ this method to monitor host and graft activity in various models of tissue regeneration. We implemented real-time bioluminescence functional imaging to analyze bone formation by following a unique protocol in which the luciferase reporter gene, driven by an osteocalcin promoter, is used to visualize host and graft activity during bone formation. Real-time bioluminescence functional imaging can be used to assess the "host reaction" in transgenic mice models; it can also be used to assess "graft activity" in other animals in which genetically labeled stem cells have been implanted or direct gene delivery has been applied. The suggested imaging protocol requires 25 min per sample. However, special attention must be given to the layout of the experimental design, which determines the specific activity that will be analyzed.

Intervertebral disc and facet joints are the two primary load-bearing structures of the lumbar spine, and altered loading to these structures may be associated with frontal plane spinal deviations.To determine the load on the lumbar facet joint and intervertebral disc under simulated frontal plane pelvic obliquity combined loading, an in vitro biomechanical study was conducted.An in vitro biomechanical study using a repeated-measures design was used to compare L4-L5 facet joint and intervertebral disc loading across pure moment and combined loading conditions.Eight fresh-frozen lumbosacral specimens were tested under five loading conditions: flexion/extension, lateral bending, axial rotation using pure moment bending (±10 Nm), and two additional tests investigating frontal plane pelvic obliquity and axial rotation (sacrum tilted left 5° and at 10° followed by a ±10-Nm rotation moment). Three-dimensional kinematics, facet load, and intradiscal pressures were recorded from the L4-L5 functional spinal unit.Sagittal and frontal plane loading resulted in significantly smaller facet joint forces compared with conditions implementing a rotation moment (p.05). The facet joint had the highest peak load during the 10° combined loading condition (124.0±30.2 N) and the lowest peak load in flexion (26.8±16.1 N). Intradiscal pressure was high in lateral flexion (495.6±280.9 kPa) and flexion (429.0±212.9 kPa), whereas intradiscal pressures measured in rotation (253.2±135.0 kPa) and 5° and 10° combined loading conditions were low (255.5±132.7 and 267.1±127.1 kPa, respectively).Facet loading increased during simulated pelvic obliquity in frontal and transverse planes, whereas intradiscal pressures were decreased compared with sagittal and frontal plane motions alone. Altered spinopelvic alignment may increase the loads experienced by spinal tissue, especially the facet joints.

Vertebral compression fractures (VCFs), the most common fragility fractures, account for approximately 700,000 injuries per year. Since open surgery involves morbidity and implant failure in the osteoporotic patient population, a new minimally invasive biological solution to vertebral bone repair is needed. Previously, we showed that adipose-derived stem cells (ASCs) overexpressing a BMP gene are capable of inducing spinal fusion in vivo. We hypothesized that a direct injection of ASCs, designed to transiently overexpress rhBMP6, into a vertebral bone void defect would accelerate bone regeneration. Porcine ASCs were isolated and labeled with lentiviral vectors that encode for the reporter gene luciferase (Luc) under constitutive (ubiquitin) or inductive (osteocalcin) promoters. The ASCs were first labeled with reporter genes and then nucleofected with an rhBMP6-encoding plasmid. Twenty-four hours later, bone void defects were created in the coccygeal vertebrae of nude rats. The ASC-BMP6 cells were suspended in fibrin gel (FG) and injected into the bone void. A control group was injected with FG alone. The regenerative process was monitored in vivo using microCT, and cell survival and differentiation were monitored using tissue specific reporter genes and bioluminescence imaging (BLI). The surgically treated vertebrae were harvested after 12 weeks and subjected to histological and immunohistochemical (against porcine vimentin) analyses. In vivo BLI detected Luc-expressing cells at the implantation site over a 12-week period. Beginning 2 weeks postoperatively, considerable defect repair was observed in the group treated with ASC-BMP6 cells. The rate of bone formation in the stem cell-treated group was two times faster than that in the FG-treated group, and bone volume at the end point was 2-fold compared to the control group. Twelve weeks after cell injection the bone volume within the void reached the volume measured in native vertebrae. Immunostaining against porcine vimentin indicated that the ASC-BMP6 cells contributed to new bone formation. Here we show the potential of injections of BMP-modified ASCs to repair vertebral bone defects in a rat model. Our results could pave the way to a novel approach for the biological treatment of traumatic and osteoporosis-related vertebral bone injuries.

BACKGROUND There is an increased incidence of fractures in untreated adjacent vertebrae after vertebroplasty. OBJECTIVE To introduce unconstrained 6 degrees of freedom biomechanical testing to investigate whether vertebroplasty lowered the fracture strength of adjacent untreated vertebrae under physiological loading conditions and to describe the observed fracture pattern. METHODS Three-level spinal segments (T10-12 and L1-3) from 6 spines were tested under unconstrained axial compression in which shear forces and torque were minimized using a 6-degrees of freedom robotic arm. Fracture initiation loads and ultimate failure loads of lumbar segments were predicted from the corresponding thoracic segments by assuming constant fracture stress along the spinal column. The predicted values were compared with postvertebroplasty experimental values of the lumbar spine segments. Plain radiographs were taken at 600-N increments to record the developing fracture pattern. RESULTS All 6 vertebroplasty group specimens experienced reductions in fracture strengths ranging from 27.4% to 47.6% with an average decrease of 32.6% (P < .002) and reductions in ultimate failure load ranging from 1.6% to 47.3%, with an average decrease of 34.7% (P < .003) compared with predicted values from the nonvertebroplasty group. In all vertebroplasty group specimens, the superior and inferior endplates of the untreated middle vertebral body (L2) were deflected, whereas 5 of the 6 nonvertebroplasty group specimens did not show any evidence of endplate deflection. CONCLUSION Vertebroplasty altered the load transfer along the anterior spinal column, thereby statistically significantly increasing fracture risk and ultimate failure load of the untreated adjacent vertebrae. The radiographic findings support the endplate deflection fracture mechanism as the cause of adjacent fractures after vertebroplasty.

Deficiencies The lumbar facet joints are the major load bearing elements in the lumbar spine posterior column and they are considered one of the anatomical structures that can cause low back pain. They also contribute to the amount and type of lumbar spine motion.Copyright © 2010 by ASME