Our results demonstrate, for the first time, that human AF and NP cells constitutively express NGF protein and mRNA, and that the proinflammatory cytokines IL-1beta and TNF-alpha stimulate the production of NGF. The precise role of NGF produced by IVD cells in the generation of discogenic pain or on the metabolism of IVD cells, especially under certain physiologic conditions in which cytokines are up-regulated, needs to be clarified in future experimentation.

Objective. Interleukin-1 receptor antagonist (IL1Ra) has been evaluated for the intraarticular treatment of osteoarthritis. Such administration of proteins may have limited utility because of their rapid clearance and short half-life in the joint. The fusion of a drug to elastin-like polypeptides (ELPs) promotes the formation of aggregating particles that form a "drug depot" at physiologic temperatures, a phenomenon intended to prolong the presence of the drug. The purpose of this study was to develop an injectable drug depot composed of IL-1Ra and ELP domains and to evaluate the properties and bioactivity of the recombinant ELP-IL-1Ra fusion protein.Methods. Fusion proteins between IL-1Ra and 2 distinct sequences and molecular weights of ELP were overexpressed in Escherichia coli. Environmental sensitivity was demonstrated by turbidity and dynamic light scattering as a function of temperature. IL-1Ra domain activity was evaluated by surface plasmon resonance, and in vitro antagonism of IL-1-mediated lymphocyte and thymocyte proliferation, as well as IL-1-induced tumor necrosis factor ␣ (TNF␣) expression and matrix metalloproteinase 3 (MMP-3) and ADAMTS-4 messenger RNA expression in human intervertebral disc fibrochondrocytes. IL-1Ra immunoreactivity was assessed before and after proteolytic degradation of the ELP partner.Results. Both fusion proteins underwent supramolecular aggregation at subphysiologic temperatures and slowly resolubilized at 37°C. Interaction with IL-1 receptor was slower in association but equivalent in dissociation as compared with the commercial antagonist. Anti-IL-1 activity was demonstrated by inhibition of lymphocyte and thymocyte proliferation and by decreased TNF␣ expression and ADAMTS-4 and MMP-3 transcription by fibrochondrocytes. ELP domain proteolysis liberated a peptide of comparable size and immunoreactivity as the commercial IL-1Ra. This peptide was more bioactive against lymphocyte proliferation, nearly equivalent to the commercial antagonist.Conclusion. The ELP-IL-1Ra fusion protein proved to retain the characteristic ELP inverse phasetransitioning behavior as well as the bioactivity of the IL-1Ra domain. This technology represents a novel drug carrier designed to prolong the presence of bioactive peptides following intraarticular delivery.Interleukin-1 (IL-1) has been implicated as a mediator of anabolic and catabolic processes in the progression of osteoarthritis (OA). IL-1 is a 17-kd protein that stimulates both synoviocytes and chondrocytes to secrete proteases and other

Platelet-rich plasma was effective in stimulating cell proliferation and extracellular matrix metabolism. The response to platelet-rich plasma was greater in the case of anulus fibrosus cells than of nucleuspulposus cells. The local administration of platelet-rich plasma might stimulate intervertebral disc repair. In addition, given the risks of using animal serum for tissue engineering, autologous blood may gain favor as a source of growth factors and serum supplements needed for stimulating cells to engineer intervertebral disc tissues.

Cerebral palsy (CP) is the most common cause of pediatric neurodevelopmental and physical disability in the United States. It is defined as a group of motor disorders caused by a nonprogressive perinatal insult to the brain. Although the brain lesion is nonprogressive, there is a progressive, lifelong impact on skeletal muscles, which are shorter, spastic, and may develop debilitating contractures. Satellite cells are resident muscle stem cells that are indispensable for postnatal growth and regeneration of skeletal muscles. Here we measured the myogenic potential of satellite cells isolated from contractured muscles in children with CP. When compared with typically developing (TD) children, satellite cell-derived myoblasts from CP differentiated more slowly (slope: 0.013 (SD 0.013) CP vs. 0.091 (SD 0.024) TD over 24 h, P < 0.001) and fused less (fusion index: 21.3 (SD 8.6) CP vs. 81.3 (SD 7.7) TD after 48 h, P < 0.001) after exposure to low-serum conditions that stimulated myotube formation. This impairment was associated with downregulation of several markers important for myoblast fusion and myotube formation, including DNA methylation-dependent inhibition of promyogenic integrin-β 1D (ITGB1D) protein expression levels (-50% at 42 h), and ~25% loss of integrin-mediated focal adhesion kinase phosphorylation. The cytidine analog 5-Azacytidine (5-AZA), a demethylating agent, restored ITGB1D levels and promoted myogenesis in CP cultures. Our data demonstrate that muscle contractures in CP are associated with loss of satellite cell myogenic potential that is dependent on DNA methylation patterns affecting expression of genetic programs associated with muscle stem cell differentiation and muscle fiber formation.

IntroductionIntervertebral disc (IVD) degeneration is associated with proteolytic degradation of the extracellular matrix, and its repair requires both the production of extracellular matrix and the downregulation of proteinase activity. These properties are associated with several growth factors. However, the use of growth factors in clinical practice is limited by their high cost. This cost can be circumvented using synthetic peptides, such as Link N, which can stimulate the synthesis of proteoglycan and collagen by IVD cells in vitro. The purpose of the present study was to evaluate the effect of Link N in vivo in a rabbit model of IVD degeneration.MethodsNew Zealand white rabbits received annular puncture in two lumbar discs. Two weeks after puncture, both punctured discs of each rabbit were injected with either Link N or saline. After 2 weeks, nine rabbits were euthanized and the annulus fibrosus (AF) and nucleus pulposus (NP) of Link N-injected and saline-injected IVDs were removed and used to prepare total RNA. Following reverse transcription, quantitative PCR was performed for aggrecan, COL2A1, COL1A1, ADAMTS-4, ADAMTS-5 and MMP-3. After 12 weeks, 19 rabbits were euthanized and the injected IVDs were removed for biochemical and histological analysis. Proteinase K digests were analyzed for DNA and sulfated glycosaminoglycan content. Disc height was monitored radiographically biweekly.ResultsFollowing needle puncture, disc height decreased by about 25% over 2 weeks, and was partially restored by Link N injection. Puncture of the IVD resulted in a trend towards decreased proteoglycan content in both the NP and AF, and a trend towards partial restoration following Link N injection, although under the time course used this did not achieve statistical significance. Link N did not alter the DNA content of the discs. Link N injection led to a significant increase in aggrecan gene expression and a significant decrease in proteinase gene expression in both the NP and AF, when compared with saline alone.ConclusionsWhen administered to the degenerate disc in vivo, Link N stimulated aggrecan gene expression and downregulated metalloproteinase expression, and there was a trend towards increased proteoglycan content of the disc, in both the NP and AF. These are features needed for any agent designed to stimulate disc repair. In principle, therefore, Link N supplementation could be an option for treating disc degeneration.