Background Chronic low back pain can be associated with the pathological ingrowth of blood vessels and nerves into intervertebral discs (IVDs). The notochord patterns the IVD during development and is a source of anti-angiogenic soluble factors such as Noggin and Chondroitin sulfate (CS). These factors may form the basis for a new minimally invasive strategy to target angiogenesis in the IVD. Objective To examine the anti-angiogenic potential of soluble factors from notochordal cells (NCs) and candidates Noggin and CS under healthy culture conditions and in the presence of pro-inflammatory mediators. Design NC conditioned media (NCCM) was generated from porcine NC-rich nucleus pulposus tissue. To assess the effects of NCCM, CS and Noggin on angiogenesis, cell invasion and tubular formation assays were performed using human umbilical vein endothelial cells (HUVECs) ± tumor necrosis factor alpha (TNFα [10 ng/ml]). vascular endothelial growth factor (VEGF)-A, MMP-7, interleukin-6 (IL-6) and IL-8 mRNA levels were assessed using qRT-PCR. Results NCCM (10 & 100%), CS (10 and 100 μg) and Noggin (10 and 100 ng) significantly decreased cell invasion of HUVECs with and without TNFα. NCCM 10% and Noggin 10 ng inhibited tubular formation with and without TNFα and CS 100 μg inhibited tubules in Basal conditions whereas CS 10 μg inhibited tubules with TNFα. NCCM significantly decreased VEGF-A, MMP-7 and IL-6 mRNA levels in HUVECs with and without TNFα. CS and Noggin had no effects on gene expression. Conclusions We provide the first evidence that soluble factors from NCs can inhibit angiogenesis by suppressing VEGF signaling. Notochordal-derived ligands are a promising minimally invasive strategy targeting neurovascular ingrowth and pain in the degenerated IVD.

IntroductionNotochordal cells (NCs) pattern aneural and avascular intervertebral discs (IVDs), and their disappearance, is associated with onset of IVD degeneration. This study induced and characterized the maturation of nucleus pulposus (NP) tissue from a gelatinous NC-rich structure to a matrix-rich structure populated by small NP cells using dynamic pressurization in an ex vivo culture model, and also identified soluble factors from NCs with therapeutic potential.MethodsPorcine NC-rich NP tissue was cultured and loaded with hydrostatic pressure (0.5 to 2 MPa at 0.1 Hz for 2 hours) either Daily, for 1 Dose, or Control (no pressurization) groups for up to eight days. Cell phenotype and tissue maturation was characterized with measurements of cell viability, cytomorphology, nitric oxide, metabolic activity, matrix composition, gene expression, and proteomics.ResultsDaily pressurization induced transition of NCs to small NP cells with 73.8%, 44%, and 28% NCs for Control, 1 Dose and Daily groups, respectively (P < 0.0002) and no relevant cell death. Dynamic loading matured NP tissue by significantly increasing metabolic activity and accumulating Safranin-O-stained matrix. Load-induced maturation was also apparent from the significantly decreased glycolytic, cytoskeletal (Vimentin) and stress-inducible (HSP70) proteins assessed with proteomics. Loading increased the production of bioactive proteins Sonic Hedgehog (SHH) and Noggin, and maintained Semaphorin3A (Sema3A).DiscussionNP tissue maturation was induced from dynamic hydrostatic pressurization in a controlled ex vivo environment without influence from systemic effects or surrounding structures. NCs transitioned into small nonvacuolated NP cells probably via differentiation as evidenced by high cell viability, lack of nitric oxide and downregulation of stress-inducible and cytoskeletal proteins. SHH, Sema3A, and Noggin, which have patterning and neurovascular-inhibiting properties, were produced in both notochordal and matured porcine NP. Results therefore provide an important piece of evidence suggesting the transition of NCs to small NP cells is a natural part of aging and not the initiation of degeneration. Bioactive candidates identified from young porcine IVDs may be isolated and harnessed for therapies to target discogenic back pain.

BACKGROUND CONTEXT Painful human intervertebral discs (IVDs) exhibit nerve growth deep into the IVD. Current treatments for discogenic back pain do not address the underlying mechanisms propagating pain and are often highly invasive or only offer temporary symptom relief. The notochord produces factors during development that pattern the spine and inhibit growth of dorsal root ganglia (DRG) axons into the IVD. We hypothesize that notochordal cell (NC) conditioned medium (NCCM) includes soluble factors capable of inhibiting neurite growth and may represent a future therapeutic target. PURPOSE To test if NCCM can inhibit neurite growth and to determine if NC-derived Glycosaminoglycans (GAGs) are necessary candidates for this inhibition. STUDY DESIGN Human neuroblastoma (SH-SY5Y) cells and rat dorsal root ganglion (DRGs) cells were treated with NCCM in 2D culture in vitro and digestion and mechanistic studies determined if specific GAGs were responsible for inhibitory effects. METHODS NCCM was generated from porcine nucleus pulposus tissue that was cultured in DMEM for 4 days. A dose study was performed using SH-SY5Y cells that were seeded in basal medium for 24hrs and neurite outgrowth and cell viability were assessed following treatment with basal or NCCM (10% and 100%) media for 48hrs. GAGs from NCCM were characterized using multiple digestions and liquid chromatography mass spectroscopy (LC-MS). Neurite growth was assessed on both SH-SY5Y and DRG cells following treatment with NCCM with and without GAG digestion. RESULTS NCCM significantly inhibited neurite outgrowth from SH-SY5Y cells compared to Basal controls without dose or cytotoxic effects; % neurite expressing cells were 39.0±2.9, 27.3±3.6 & 30.2±2.7, and mean neurite length was 60.3±3.5μm, 50.8±2.4μm, 53.2±3.7μm for Basal, 10% NCCM & 100% NCCM, respectively. Digestions and LC-MS determined that Chondroitin-6-Sulfate was the major GAG chain in NCCM. Neurite growth from SH-SY5Y and DRG cells was not inhibited when cells were treated with NCCM with digested chondroitin sulfate (CS). CONCLUSIONS Soluble factors derived from NCCM were capable of inhibiting neurite outgrowth in multiple neural cell types without any negative effects on cell viability. Cleavage of GAGs via digestion was necessary to reverse the neurite inhibition capacity of NCCM. We conclude that intact GAGs such as CS secreted from NCs are potential candidates that could be useful to reduce neurite growth in painful IVDs.

An understanding of the processes that occur during development of the intervertebral disk can help inform therapeutic strategies for discogenic pain. This article reviews the literature to identify candidates that are found in or derived from the notochord or notochordal cells and evaluates the theory that such factors could be isolated and used as biologics to target the structural disruption, inflammation, and neurovascular ingrowth often associated with discogenic back pain. A systematic review using PubMed was performed with a primary search using keywords “(notochordal OR notochord) And (nerves OR blood vessels OR SHH OR chondroitin sulfate OR notch OR CTGF) NOT chordoma.” Secondary searches involved keywords associated with the intervertebral disk and pain. Several potential therapeutic candidates from the notochord and their possible targets were identified. Studies are needed to further identify candidates, explore mechanisms for effect, and to validate the theory that these candidates can promote structural restoration and limit or inhibit neurovascular ingrowth using in vivo studies.

Introduction Satisfaction with breast reconstruction is thought to be greatest among patients who complete nipple and areolar-complex (NAC) reconstruction. Anecdotally, many patients are known to decline NAC reconstruction. The authors aimed to characterize the epidemiology of and factors associated with incomplete breast reconstruction. Methods Breast reconstruction patients with follow-up in a single institution's electronic medical record system were reviewed. Chi-squared and independent t-tests were used to identify variables associated with lack of NAC reconstruction; associated variables (p<0.05) were used to build a binary logistic regression. Results Four hundred thirty-three patients were reviewed. Reconstructions consisted of an average of 4.0±2.0 procedures over 503 (range 2-3,652) days. One hundred twelve patients had NAC reconstruction or tattooing (25.9%) and 73 (17.6%) had both – 226 women (54.6%) had neither. On multivariate analysis, a history of any implant removal was associated with a 93.4% decreased chance of NAC reconstruction (p=0.002), while prophylactic or early stage mastectomy was associated with 52.9% increased chances of NAC reconstruction (p=0.009). Conclusions Over half of the present cohort did not complete any NAC reconstruction. Patients with later-stage cancer and a history of implant removal were less likely to have NAC reconstruction. The high prevalence of incomplete reconstructions suggests that the classical definition of breast reconstruction completion as requiring NAC reconstruction may be outdated or not applicable to all populations. Instead, “completion” should be considered a subjective determination varying between patients.