A Single Injection of NTG-101 Reduces the Expression of Pain-Related Neurotrophins in a Canine Model of Degenerative Disc Disease.

BACKGROUND: Tissue sources of pain emanating from degenerative discs remains incompletely understood. Canine intervertebral discs (IVDs) were needle puncture injured, 4-weeks later injected with either phosphate-buffered saline (PBS) or NTG-101, harvested after an additional fourteen weeks and then histologically evaluated for the expression of NGFr, BDNF, TrkB and CALCRL proteins. Quantification was performed using the HALO automated cell-counting scoring platform. Immunohistochemical analysis was also performed on human IVD tissue samples obtained from spinal surgery. Immunohistochemical analysis and quantification of neurotrophins and neuropeptides was performed using an in vivo canine model of degenerative disc disease and human degenerative disc tissue sections. Discs injected with NTG-101 showed significantly lower levels of Nerve Growth Factor receptor (NGFr/TrkA, p = 0.0001), BDNF (p = 0.009), TrkB (p = 0.002) and CALCRL (p = 0.008) relative to PBS injections. Human IVD tissue obtained from spinal surgery due to painful DDD show robust expression of NGFr, BDNF, TrkB and CALCRL proteins. A single intradiscal injection of NTG-101 significantly inhibits the expression of NGFr, BDNF, TrkB and CALCRL proteins in degenerative canine IVDs. These results strongly suggest that NTG-101 inhibits the development of neurotrophins that are strongly associated with painful degenerative disc disease and may have profound effects upon the management of patients living with discogenic pain.

Spheroid-Based Tissue Engineering Strategies for Regeneration of the Intervertebral Disc.

Degenerative disc disease, a painful pathology of the intervertebral disc (IVD), often causes disability and reduces quality of life. Although regenerative cell-based strategies have shown promise in clinical trials, none have been widely adopted clinically. Recent developments demonstrated that spheroid-based approaches might help overcome challenges associated with cell-based IVD therapies. Spheroids are three-dimensional multicellular aggregates with architecture that enables the cells to differentiate and synthesize endogenous ECM, promotes cell-ECM interactions, enhances adhesion, and protects cells from harsh conditions. Spheroids could be applied in the IVD both in scaffold-free and scaffold-based configurations, possibly providing advantages over cell suspensions. This review highlights areas of future research in spheroid-based regeneration of nucleus pulposus (NP) and annulus fibrosus (AF). We also discuss cell sources and methods for spheroid fabrication and characterization, mechanisms related to spheroid fusion, as well as enhancement of spheroid performance in the context of the IVD microenvironment.

Developmentally inspired programming of adult human mesenchymal stromal cells toward stable chondrogenesis.

It is generally accepted that adult human bone marrow-derived mesenchymal stromal cells (hMSCs) are default committed toward osteogenesis. Even when induced to chondrogenesis, hMSCs typically form hypertrophic cartilage that undergoes endochondral ossification. Because embryonic mesenchyme is obviously competent to generate phenotypically stable cartilage, it is questioned whether there is a correspondence between mesenchymal progenitor compartments during development and in adulthood. Here we tested whether forcing specific early events of articular cartilage development can program hMSC fate toward stable chondrogenesis. Inspired by recent findings that spatial restriction of bone morphogenetic protein (BMP) signaling guides embryonic progenitors toward articular cartilage formation, we hypothesized that selective inhibition of BMP drives the phenotypic stability of hMSC-derived chondrocytes. Two BMP type I receptor-biased kinase inhibitors were screened in a microfluidic platform for their time- and dose-dependent effect on hMSC chondrogenesis. The different receptor selectivity profile of tested compounds allowed demonstration that transient blockade of both ALK2 and ALK3 receptors, while permissive to hMSC cartilage formation, is necessary and sufficient to maintain a stable chondrocyte phenotype. Remarkably, even upon compound removal, hMSCs were no longer competent to undergo hypertrophy in vitro and endochondral ossification in vivo, indicating the onset of a constitutive change. Our findings demonstrate that adult hMSCs effectively share properties of embryonic mesenchyme in the formation of transient but also of stable cartilage. This opens potential pharmacological strategies to articular cartilage regeneration and more broadly indicates the relevance of developmentally inspired protocols to control the fate of adult progenitor cell systems.

Effectiveness of the addition of Lidocaine to a hemostatic, bioresorbable putty in the treatment of iliac crest donor site pain.

BACKGROUND: The harvest of iliac crest bone grafts (ICBG) is associated with relevant donor site pain, but may be lowered by the application of lidocaine loaded on biodegradable, hemostatic putty for sustained local analgesic release. The goal of this double-blind controlled trial was to assess the efficacy of adding lidocaine to a hemostatic putty (Orthostat ™) to treat donor site pain following harvest of ICBG in foot and ankle procedures. METHODS: After ICBG harvest during a foot and ankle procedure, the resulting bone defect was either filled with Orthostat™ (n = 7) or with the same hemostatic putty loaded with lidocaine (Orthostat-L™, n = 7). During the first 72 postoperative hours, donor site and surgical site pain were managed by patient controlled morphine delivery and a peripheral nerve block. Donor site pain was periodically quantified on a Visual Analog (VAS) and a Wong Baker FACES scale. Pain scores were plotted over time to calculate the area under the curve (AUC) to quantify the overall pain experienced in specific time intervals. RESULTS: Orthostat-L™ significantly reduced donor site pain over the first 12 hours postoperatively as evidenced by a significant decrease of the AUC in both VAS (p = 0.0366) and Wong Baker FACES pain score plots (p = 0.0024). Cumulated morphine uses were not significantly decreased with Orthostat-L™. CONCLUSION: The addition of lidocaine to a hemostatic putty offers a significant ICBG donor site pain reduction over the first 12 postoperative hours. TRIAL REGISTRATION: ClinicalTrials.gov NCT01504035. Registered January 2nd 2012.

Fibroblast growth factor-2 maintains a niche-dependent population of self-renewing highly potent non-adherent mesenchymal progenitors through FGFR2c.

Bone marrow (BM) mesenchymal stem/stromal cells (MSC) are a heterogeneous population of multipotent progenitors currently under investigation for a variety of applications in regenerative medicine. While self-renewal of stem cells in different tissues has been demonstrated to be regulated by specialized microenvironments called niches, it is still unclear whether a self-renewing niche also exists for MSC. Here, we show that primary human BM cultures contain a population of intrinsically non-adherent mesenchymal progenitors (NAMP) with features of more primitive progenitors than the initially adhering colony-forming units-fibroblast (CFU-f). In fact, NAMP could generate an adherent progeny: (a) enriched with early mesenchymal populations (CD146+, SSEA-1+, and SSEA-4+); (b) with significantly greater proliferation and multilineage differentiation potential in vitro; and (c) capable of threefold greater bone formation in vivo than the corresponding CFU-f. Upon serial replating, NAMP were able to regenerate and expand in suspension as non-adherent clonogenic progenitors, while also giving rise to an adherent progeny. This took place at the cost of a gradual loss of proliferative potential, shown by a reduction in colony size, which could be completely prevented when NAMP were expanded on the initially adhering BM fraction. Mechanistically, we found that NAMP crucially depend on fibroblast growth factor (FGF)-2 signaling through FGFR2c for their survival and expansion. Furthermore, NAMP maintenance depends at least in part on humoral signals distinct from FGF-2. In conclusion, our data show a niche/progenitor organization in vitro, in which the BM adherent fraction provides a self-renewing microenvironment for primitive NAMP.

Substitutes of structural and non-structural autologous bone grafts in hindfoot arthrodeses and osteotomies: a systematic review.

BACKGROUND: Structural and non-structural substitutes of autologous bone grafts are frequently used in hindfoot arthrodeses and osteotomies. However, their efficacy is unclear.The primary goal of this systematic review was to compare autologous bone grafts with structural and non-structural substitutes regarding the odds of union in hindfoot arthrodeses and osteotomies. METHODS: The Medline and EMBASE and Cochrane databases were searched for relevant randomized and non-randomized prospective studies as well as retrospective comparative chart reviews. RESULTS: 10 studies which comprised 928 hindfoot arthrodeses and osteotomies met the inclusion criteria for this systematic review. The quality of the retrieved studies was low due to small samples sizes and confounding variables. The pooled random effect odds for union were 12.8 (95% CI 12.7 to 12.9) for structural allografts, 5.7 (95% CI 5.5 to 6.0) for cortical autologous grafts, 7.3 (95% CI 6.0 to 8.6) for cancellous allografts and 6.0 (95% CI 5.7 to 6.4) for cancellous autologous grafts. In individual studies, the odds of union in hindfoot arthrodeses achieved with cancellous autologous grafts was similar to those achieved with demineralised bone matrix or platelet derived growth factor augmented ceramic granules. CONCLUSION: Our results suggest an equivalent incorporation of structural allografts as compared to autologous grafts in hindfoot arthrodeses and osteotomies. There is a need for prospective randomized trials to further clarify the role of substitutes of autologous bone grafts in hindfoot surgery.

Human 3D nucleus pulposus microtissue model to evaluate the potential of pre-conditioned nasal chondrocytes for the repair of degenerated intervertebral disc.

Introduction: An in vitro model that appropriately recapitulates the degenerative disc disease (DDD) microenvironment is needed to explore clinically relevant cell-based therapeutic strategies for early-stage degenerative disc disease. We developed an advanced 3D nucleus pulposus (NP) microtissues (µT) model generated with cells isolated from human degenerating NP tissue (Pfirrmann grade: 2-3), which were exposed to hypoxia, low glucose, acidity and low-grade inflammation. This model was then used to test the performance of nasal chondrocytes (NC) suspension or spheroids (NCS) after pre-conditioning with drugs known to exert anti-inflammatory or anabolic activities. Methods: NPµTs were formed by i) spheroids generated with NP cells (NPS) alone or in combination with ii) NCS or iii) NC suspension and cultured in healthy or degenerative disc disease condition. Anti-inflammatory and anabolic drugs (amiloride, celecoxib, metformin, IL-1Ra, GDF-5) were used for pre-conditioning of NC/NCS. The effects of pre-conditioning were tested in 2D, 3D, and degenerative NPµT model. Histological, biochemical, and gene expression analysis were performed to assess matrix content (glycosaminoglycans, type I and II collagen), production and release of inflammatory/catabolic factors (IL-6, IL-8, MMP-3, MMP-13) and cell viability (cleaved caspase 3). Results: The degenerative NPµT contained less glycosaminoglycans, collagens, and released higher levels of IL-8 compared to the healthy NPµT. In the degenerative NPµT, NCS performed superior compared to NC cell suspension but still showed lower viability. Among the different compounds tested, only IL-1Ra pre-conditioning inhibited the expression of inflammatory/catabolic mediators and promoted glycosaminoglycan accumulation in NC/NCS in DDD microenvironment. In degenerative NPµT model, preconditioning of NCS with IL-1Ra also provided superior anti-inflammatory/catabolic activity compared to non-preconditioned NCS. Conclusion: The degenerative NPµT model is suitable to study the responses of therapeutic cells to microenvironment mimicking early-stage degenerative disc disease. In particular, we showed that NC in spheroidal organization as compared to NC cell suspension exhibited superior regenerative performance and that IL-1Ra pre-conditioning of NCS could further improve their ability to counteract inflammation/catabolism and support new matrix production within harsh degenerative disc disease microenvironment. Studies in an orthotopic in vivo model are necessary to assess the clinical relevance of our findings in the context of IVD repair.

Intraoperative engineering of osteogenic grafts combining freshly harvested, human adipose-derived cells and physiological doses of bone morphogenetic protein-2.

Engineered osteogenic constructs for bone repair typically involve complex and costly processes for cell expansion. Adipose tissue includes mesenchymal precursors in large amounts, in principle allowing for an intraoperative production of osteogenic grafts and their immediate implantation. However, stromal vascular fraction (SVF) cells from adipose tissue were reported to require a molecular trigger to differentiate into functional osteoblasts. The present study tested whether physiological doses of recombinant human BMP-2 (rhBMP-2) could induce freshly harvested human SVF cells to generate ectopic bone tissue. Enzymatically dissociated SVF cells from 7 healthy donors (1 x 10(6) or 4 x 10(6)) were immediately embedded in a fibrin gel with or without 250 ng rhBMP-2, mixed with porous silicated calcium-phosphate granules (Actifuse(®), Apatech) (final construct size: 0.1 cm(3)) and implanted ectopically for eight weeks in nude mice. In the presence of rhBMP-2, SVF cells not only supported but directly contributed to the formation of bone ossicles, which were not observed in control cell-free, rhBMP-2 loaded implants. In vitro analysis indicated that rhBMP-2 did not involve an increase in the percentage of SVF cells recruited to the osteogenic lineage, but rather induced a stimulation of the osteoblastic differentiation of the committed progenitors. These findings confirm the feasibility of generating fully osteogenic grafts using an easily accessible autologous cell source and low amounts of rhBMP-2, in a timing compatible with an intraoperative schedule. The study warrants further investigation at an orthotopic site of implantation, where the delivery of rhBMP-2 could be bypassed thanks to the properties of the local milieu.

Computed tomography osteoabsorptiometry for imaging of degenerative disc disease.

BACKGROUND: Lower back pain is a common condition with significant morbidity and economic impact. The pathophysiology is poorly understood but is in part attributable to degenerative disc disease (DDD). The healthy intervertebral disc ensures spine functionality by transferring the perceived load to the caudally adjacent vertebrae. The exposure to recurring mechanical load is mirrored in the mineralization pattern of the subchondral bone plate (SBP), where increased bone density is a sign of repetitive localized high stress. Computed tomography -osteoabsorptiometry (CT-OAM) is a technique based on conventional CT scans that displays the mineral density distribution in the SBP as a surface-color map. The objective of this study was to measure and analyze the SBP mineral density patterns of healthy lumbar intervertebral disc (IVDs) and those suffering DDD using CT-OAM densitograms. These findings should provide in vitro insight into the long-term morphological properties of the IVD and how these differ in the state of disc degeneration. METHODS: The CT-data sets of spines from 17 healthy individuals and 18 patients displaying DDD in the lumbar spine were acquired. Individual vertebrae of both cohorts were 3D reconstructed, processed using image analysis software, and compared to one another. Maximum intensity projection of the subchondral mineralization provided surface densitograms of the SBP. The relative calcium concentration, the local maxima of mineralization, and a mean surface projection of level-defined SBPs were calculated from the densitogram and statistically compared. RESULTS: The inferior SBP, adjacent to degenerating disc, display an 18-41 % higher relative calcium concentration than their healthy counterparts. In the opposing superior SBPs the relative calcium content is significantly increased. Whereas it is reasonably consistent for L1-L3 (L1: 132 %, L2: 127 %, L3: 120 %), the increase grows in caudal direction (L4: 131 %, L5: 148 %, S1: 152 %). Furthermore, a change in the areal distribution of excessive mineralization can be differentiated between healthy and diseased motion segments. CONCLUSIONS: The acquired data provide in vitro proof of the mechanical and anatomical properties of the SBP in relation to the state of disc degeneration. In conjunction with the diagnostic use of CT-osteoabsorptiometry, our data provide a basis for a non-invasive and sensitive technique that correlates with disc functionality. This could be promising in various cases, from early identification of early stages of DDD, tracking disease progression, and assessing the repercussions of surgical procedures or experimental therapies.

Intervertebral Disc-on-a-Chip as Advanced In Vitro Model for Mechanobiology Research and Drug Testing: A Review and Perspective.

Discogenic back pain is one of the most diffused musculoskeletal pathologies and a hurdle to a good quality of life for millions of people. Existing therapeutic options are exclusively directed at reducing symptoms, not at targeting the underlying, still poorly understood, degenerative processes. Common intervertebral disc (IVD) disease models still do not fully replicate the course of degenerative IVD disease. Advanced disease models that incorporate mechanical loading are needed to investigate pathological causes and processes, as well as to identify therapeutic targets. Organs-on-chip (OoC) are microfluidic-based devices that aim at recapitulating tissue functions in vitro by introducing key features of the tissue microenvironment (e.g., 3D architecture, soluble signals and mechanical conditioning). In this review we analyze and depict existing OoC platforms used to investigate pathological alterations of IVD cells/tissues and discuss their benefits and limitations. Starting from the consideration that mechanobiology plays a pivotal role in both IVD homeostasis and degeneration, we then focus on OoC settings enabling to recapitulate physiological or aberrant mechanical loading, in conjunction with other relevant features (such as inflammation). Finally, we propose our view on design criteria for IVD-on-a-chip systems, offering a future perspective to model IVD mechanobiology.

Nose to Spine: spheroids generated by human nasal chondrocytes for scaffold-free nucleus pulposus augmentation.

Cell-based strategies for nucleus pulposus (NP) regeneration that adequately support the engraftment and functionality of therapeutic cells are still lacking. This study explores a scaffold-free approach for NP repair, which is based on spheroids derived from human nasal chondrocytes (NC), a resilient cell type with robust cartilage-regenerative capacity. We generated NC spheroids (NCS) in two types of medium (growth or chondrogenic) and analyzed their applicability for NP repair with regard to injectability, biomechanical and biochemical attributes, and integration potential in conditions simulating degenerative disc disease (DDD). NCS engineered in both media were compatible with a typical spinal needle in terms of size (lower than 600µm), shape (roundness greater than 0.8), and injectability (no changes in morphology and catabolic gene expression after passing through the needle). While growth medium ensured stable elastic modulus (E) at 5 kPa, chondrogenic medium time-dependently increased E of NCS, in correlation with gene/protein expression of collagen. Notably, DDD-mimicking conditions did not impair NCS viability nor NCS fusion with NP spheroids simulating degenerated NP in vitro. To assess the feasibility of this approach, NCS were injected into an ex vivo-cultured bovine intervertebral disc (IVD) without damage using a spinal needle. In conclusion, our data indicated that NC cultured as spheroids can be compatible with strategies for minimally invasive NP repair in terms of injectability, tuneability, biomechanical features, and resilience. Future studies will address the capacity of NCS to integrate within degenerated NP under long-term loading conditions. STATEMENT OF SIGNIFICANCE: Current regenerative strategies still do not sufficiently support the engraftment of therapeutic cells in the nucleus pulposus (NP). We present an injectable approach based on spheroids derived from nasal chondrocytes (NC), a resilient cell type with robust cartilage-regenerative capacity. NC spheroids (NCS) generated with their own matrix and demonstrated injectability, tuneability of biomechanical/biochemical attributes, and integration potential in conditions simulating degenerative disc disease. To our knowledge, this is the first study that explored an injectable spheroid-based scaffold-free approach, which showed potential to support the adhesion and viability of therapeutic cells in degenerated NP. The provided information can be of substantial interest to a wide audience, including biomaterial scientists, biomedical engineers, biologists and medical researchers.

Efficacy and safety of VEPTR instrumentation for progressive spine deformities in young children without rib fusions.

This retrospective study analyses 23 children treated with vertical expandable prosthetic titanium rib (VEPTR) for correction of non-congenital early onset spine deformities. After the index procedure (IP), the device was lengthened at 6-month intervals. The average (av) age at the time of IP was 6.5 years (1.11-10.5). The av follow-up time was 3.6 years (2-5.8). Diagnosis included 1 early onset idiopathic scoliosis, 11 neuromuscular, 2 post-thoracotomy scoliosis, 1 Sprengel deformity, 2 hyperkyphosis, 1 myopathy and 5 syndromic. Surgeries (187) included 23 IPs, av 6.5 (4-10) device expansions per patient (149) and 15 unplanned surgeries. 23 complications (0.13 per surgery) included 10 skin sloughs, 5 implant dislocations, 2 rod breakages and 6 infections. Coronal Cobb angle was av 68 degrees (11 degrees -111 degrees ), at follow-up av 54 degrees (0 degrees -105 degrees). Pelvic obliquity was av 33 degrees (13 degrees -60 degrees ), at follow-up av 16 degrees (0 degrees -42 degrees ). T1 tilt was av 29 degrees (5 degrees -84 degrees ), two remained unchanged, the remainder improved 10 degrees -68 degrees. Sagittal plane: All but two had stable profiles, two hyperkyphosis of 110 degrees /124 degrees improved to 56 degrees /86 degrees. Space available for lung ratio was less than 90% in ten before the IP, improved in nine and deteriorated in one. Originally designed for thoracic insufficiency syndromes related to rib and vertebral anomalies, VEPTR proved to be a valuable alternative to dual growing rods for non-congenital early onset spine deformities. The complication rate was lower, the control of the sagittal plane and the pelvic obliquity was as good, but the correction of the coronal plane deformity was less than growing rods. However, VEPTR's spine-sparing approach might provoke less spontaneous spinal fusion and ease the final correction at maturity.

Challenges Toward the Identification of Predictive Markers for Human Mesenchymal Stromal Cells Chondrogenic Potential.

Human bone marrow derived mesenchymal stromal cells (BMSCs) represent a putative cell source candidate for tissue engineering-based strategies to repair cartilage and bone. However, traditional isolation of BMSCs by adhesion to plastic leads to very heterogeneous cell populations, accounting for high variability of chondrogenic differentiation outcome, both across donors and across clonally derived strains. Identification of putative surface markers able to select BMSC subpopulations with higher chondrogenic capacity (CC) and reduced variance in chondrogenic differentiation could aid the development of BMSC-based cartilage and bone regeneration approaches. With the goal to identify predictive markers for chondrogenic BMSC populations, we assessed the gene expression profile of single cell-derived clones with high and low CC. While a clustering between high and low CC clones was observed for one donor, donor-to-donor variability hampered the possibility to achieve conclusive results when different donors were considered. Nevertheless, increased NCAM1/CD56 expression correlated in clones derived from one donor with higher CC, the same trend was observed for three additional donors (even if no significance was achieved). Enriching multiclonal BMSCs for CD56+ expression led to an increase in CC, though still highly affected by donor-to-donor variability. Our study finally suggests that definition of predictive marker(s) for BMSCs chondrogenesis is challenged by the large donor heterogeneity of these cells, and by the high complexity and plasticity of the BMSCs system. Multiple pathways and external parameters may be indeed involved in determining the chondrogenic potential of BMSCs, making the identification of putative markers still an open issue. Stem Cells Translational Medicine 2019;8:194&11.

Minimum ten-year follow-up of spinal stenosis with degenerative spondylolisthesis treated with decompression and dynamic stabilization.

BACKGROUND: The Dynesys system remains the most widely implanted posterior non-fusion pedicle screw system. Various study designs used in investigations with good to excellent short- and mid-term results have been reported in the current literature. However, there is a lack of information concerning long-term outcomes following treatment for spinal stenosis with degenerative spondylolisthesis. METHODS: The aim of our study was twofold. Firstly, to assess whether the dynamic stabilization in situ with the Dynesys System without bone grafting provides enough stability to prevent progression of spondylolisthesis and secondarily to maintain significant clinical improvement in a long-term follow-up (FU). Therefore, the consecutive patients due to inclusion criterions underwent interlaminar decompression and stabilization with Dynesys instrumentation. Patients were evaluated clinically and radiologically after a minimum FU of 10 years. RESULTS: At FU, the mean low back pain (LBP) post-operatively and leg pain (LP) post-operatively on visual analog scale (VAS) and North American Spine Society (NASS) improved significantly (P<0.001) compared to preoperative examination. The mean value of NASS neurogenic symptoms (19.13% and 4.72%) and activity subscores were 23.13% and 10.74% respectively. In plain and functional radiographs the mean listhesis grade in neutral position was 11.11%, 11.8% in reclination and 11.63% in inclination. There were 17 and 8 patients with progressing degenerative osteochondrosis/listhesis at adjacent segments. CONCLUSIONS: Decompression and single and double level dynamic in situ stabilization with the Dynesys System demonstrate good clinical and radiological long-term results in elderly patients.

An In Vitro Bone Model to Investigate the Role of Triggering Receptor Expressed on Myeloid Cells-2 in Bone Homeostasis.

Triggering receptor expressed on myeloid cells-2 (TREM-2), a transmembrane receptor expressed by macrophages, microglia, and osteoclasts (OCs), plays a protective role in late-onset Alzheimer Disease (AD). To validate TREM-2 as a therapeutic target in AD, its potential secondary parallel effect on bone homeostasis should be clarified. However, animal models and monolayer cultures of human cells were shown poorly predictive of TREM-2 function in human. Therefore, this study aimed to engineer a tridimensional in vitro model using human progenitors differentiated into osteoblasts and OCs, recapitulating physiological bone homeostasis. Human bone marrow-derived mesenchymal cells were seeded and cultured under perfusion inside a collagen type I scaffold for 3 weeks, generating osteoblasts and mineralized matrix. Human peripheral blood-derived CD14+ monocytes were subsequently seeded through the generated tissue, thanks to perfusion flow, and further cultured for up to 3 weeks with an inductive medium, generating mature OCs. This culture system supported collagenous matrix deposition and resorption, allowing for the investigation of kinetic of soluble TREM-2 over the coculture time. Agonistic activation of TREM-2 in this model had no effect on OC activity or on mineralized matrix turnover. In conclusion, the engineered culture system represents a tridimensional, in vitro human bone model for drug testing and suggested no effect of TREM-2 agonist on bone resorption.

Spatially confined induction of endochondral ossification by functionalized hydrogels for ectopic engineering of osteochondral tissues.

Despite the various reported approaches to generate osteochondral composites by combination of different cell types and materials, engineering of templates with the capacity to autonomously and orderly develop into cartilage-bone bi-layered structures remains an open challenge. Here, we hypothesized that the embedding of cells inducible to endochondral ossification (i.e. bone marrow derived mesenchymal stromal cells, BMSCs) and of cells capable of robust and stable chondrogenesis (i.e. nasal chondrocytes, NCs) adjacent to each other in bi-layered hydrogels would develop directly in vivo into osteochondral tissues. Poly(ethylene glycol) (PEG) hydrogels were functionalized with TGFß3 or BMP-2, enzymatically polymerized encapsulating human BMSCs, combined with a hydrogel layer containing human NCs and ectopically implanted in nude mice without pre-culture. The BMSC-loaded layers reproducibly underwent endochondral ossification and generated ossicles containing bone and marrow. The NC-loaded layers formed cartilage tissues, which (under the influence of BMP-2 but not of TGFß3 from the neighbouring layer) remained phenotypically stable. The proposed strategy, resulting in orderly connected osteochondral composites, should be further assessed for the repair of osteoarticular defects and will be useful to model developmental processes leading to cartilage-bone interfaces.

Expansion of Bone Marrow Mesenchymal Stromal Cells in Perfused 3D Ceramic Scaffolds Enhances In Vivo Bone Formation.

Bone marrow-derived mesenchymal stromal cells (BMSC), when expanded directly within 3D ceramic scaffolds in perfusion bioreactors, more reproducibly form bone when implanted in vivo as compared to conventional expansion on 2D polystyrene dishes/flasks. Since the bioreactor-based expansion on 3D ceramic scaffolds encompasses multiple aspects that are inherently different from expansion on 2D polystyrene, we aimed to decouple the effects of specific parameters among these two model systems. We assessed the effects of the: 1) 3D scaffold vs. 2D surface; 2) ceramic vs. polystyrene materials; and 3) BMSC niche established within the ceramic pores during in vitro culture, on subsequent in vivo bone formation. While BMSC expanded on 3D polystyrene scaffolds in the bioreactor could maintain their in vivo osteogenic potential, results were similar as BMSC expanded in monolayer on 2D polystyrene, suggesting little influence of the scaffold 3D environment. Bone formation was most reproducible when BMSC are expanded on 3D ceramic, highlighting the influence of the ceramic substrate. The presence of a pre-formed niche within the scaffold pores had negligible effects on the in vivo bone formation. The results of this study allow a greater understanding of the parameters required for perfusion bioreactor-based manufacturing of osteogenic grafts for clinical applications.

Notochordal cell-derived conditioned medium protects human nucleus pulposus cells from stress-induced apoptosis.

BACKGROUND CONTEXT: Degenerative disc disease (DDD) remains without an effective therapy and presents a costly burden to society. PURPOSE: Based upon prior reports concerning the effects of notochordal cell-conditioned medium (NCCM) on disc cells, we performed a proof of principle study to determine whether NCCM could reduce cytotoxic stress-induced apoptosis in human disc nucleus pulposus (NP) cells. STUDY DESIGN/SETTING: This is an "in vitro" fundamental or basic science study. METHODS: Nucleus pulpous cells derived from 15 patients undergoing spinal surgery were treated with interleukin (IL)-1ß and Fas ligand or etoposide in the presence of NCCM. We determined pro- or antiapoptotic events using activated caspase assays and determined genomic regulation of apoptosis using polymerase chain reaction arrays validated using Western blotting methods. We interrogated cellular apoptotic regulation using JC-1 dye and flow cytometry and performed enzyme-linked immunosorbent assays to evaluate NP inflammatory cytokine secretion. RESULTS: Notochordal cell-conditioned medium inhibits cytotoxic stress-induced caspase-9 and -3/7 activities and maintains the mitochondrial membrane potential in human NP cells, thereby suppressing the intrinsic apoptotic pathway. Gene expression analysis revealed the X-linked inhibitor of apoptosis protein as a key player responsible for evading etoposide-induced apoptosis in the presence of NCCM, and we verified these data using Western blotting. Enzyme-linked immunosorbent assay results revealed distinct differences in IL-6 and IL-8 secretions by NP cells in response to etoposide in the presence of NCCM. CONCLUSIONS: Here we demonstrate for the first time that NCCM reduces cytotoxic stress-induced apoptosis in human NP cells. Soluble factors present in NCCM could be harnessed for the development of novel therapeutics for the treatment of DDD.

Preoperative and Postoperative Factors and Laboratory Values Predicting Outcome in Patients Undergoing Lumbar Fusion Surgery.

OBJECTIVE: To determine whether complications in lumbar fusion surgery could be estimated from patient factors and perioperative laboratory values. In addition, risk scores for detection of patients prone to complications were defined. METHODS: We retrospectively collected data of patients undergoing lumbar fusion surgery between 2013 and 2015. The patients were divided into group A (no complications) and group B (systemic and infectious complications within 30 days postoperatively). Patient-related factors and levels of perioperative laboratory values were compared between the groups and analyzed for possible impact on complications and length of stay (LOS) in the hospital. RESULTS: Data of 132 consecutive patients (74 women [56.1%]; median age, 68.5 years) were analyzed. Postoperative complications occurred in 29.5%. Higher postoperative creatine kinase (CK) and C-reactive protein and lower postoperative hemoglobin and thrombocyte values, as well as higher differences between preoperative and postoperative CK, C-reactive protein, and hemoglobin values were associated with postoperative complications. Among others, the combinations of advanced age and elevated body mass index (P = 0.0062, odds ratio: 3.018), or advanced age, elevated body mass index, and postoperative CK >166 U/L (P = 0.0016, odds ratio: 3.637) revealed patients with a threefold risk for complications. The combination of advanced age, American Society of Anesthesiologists score >2, and preoperative hemoglobin <12.9 g/dL was associated with a LOS of 20.3 versus 11 days (P = 0.01). CONCLUSIONS: Patients with postoperative complications and extended LOS seem to show significant differences in various perioperative laboratory values and patient factors. Perioperative risk assessments using cut-off values and risk scores may help identify patients prone to complications and extended resource use.

Notochordal cell conditioned medium (NCCM) regenerates end-stage human osteoarthritic articular chondrocytes and promotes a healthy phenotype.

BACKGROUND: Notochordal cell conditioned medium (NCCM) derived from non-chondrodystrophic dogs has pro-anabolic and anti-catabolic effects upon nucleus pulposus (NP) cells. Here, for the first time, we assessed the ability of NCCM to influence the production of extracellular matrix and inflammatory proteins by healthy and osteoarthritic human chondrocytes within engineered cartilage tissues. We hypothesized that, similar to its action on NP cells, NCCM exerts metabolic and anti-catabolic effects on human articular chondrocytes and has the potential to significantly counteract inflammatory mediators. METHODS: Chondrocytes from nine non-osteoarthritic patients and from six osteoarthritic (OA) donors at the time of total knee arthroplasty were chondro-differentiated in pellets for 2 weeks. Non-OA pellets were exposed for 72 hours to IL-1ß/TNF-α and then cultured up to 14 days in 2 % FBS-supplemented NCCM or 2 % FBS-supplemented medium (control (ctr)). OA pellets were cultured in NCCM or ctr medium without pro-inflammatory treatment. Tissues after each culture phase were analyzed biochemically (GAG/DNA), (immuno-) histologically (collagen I, II and GAG) and by Western blotting. Supernatants were analyzed by ELISA. RESULTS: Response to NCCM was age and disease dependent with healthy chondrocyte pellets (from donors >55 years of age) recovering their glycosaminoglycan (GAG) contents to baseline levels only with NCCM. OA pellets treated with NCCM significantly increased GAG content (1.8-fold) and levels of hyaluronic acid link protein (HAPLN), fibromodulin and SOX-9. The catabolic proteins (matrix metalloproteinase (MMP)-3 and MMP-13) and pro-inflammatory enzyme levels (cyclooxygenase-2 (COX-2)) were markedly reduced and there was significantly reduced secretion of pro-inflammatory chemokines (IL-6 and IL-8). CONCLUSIONS: NCCM restores cartilage matrix production of end-stage human OA chondrocytes towards a healthy phenotype and suppresses the production of inflammatory mediators. Harnessing the necessary and sufficient factors within NCCM that confers chondroprotection and regenerative effects could lead to a minimally invasive agent for treatment of degenerative and inflammatory joint diseases.