BACKGROUND CONTEXT Intradiscal biologic therapy (stem cell, gene, or growth factor) is a promising strategy for disc regeneration. However, these therapies require a rich nutrient supply, which may be limited by the permeability of the cartilage endplate (CEP). PURPOSE The goals of this research were to: 1) investigate how physiologic fluctuations in CEP permeability impact nutrient diffusion and nucleus pulposus (NP) cell survival and function; 2) identify compositional traits of the CEP matrix that coincide with reduced permeability; and 3) test if matrix modification of the CEP via enzymatic treatment with human MMP-8 can improve nutrient transport and NP cell survival. We hypothesized that deficits in CEP composition that hinder nutrient diffusion would associate with impaired NP cell survival and function, and that ameliorating those CEP compositional deficits via MMP-8 treatment would enhance nutrient diffusion and cell survival. STUDY DESIGN/SETTING This basic science study involved ex vivo analyses of cadaveric human CEP tissues, bovine NP cell culture, and biomechanical testing. METHODS Human CEP tissues harvested from six cadaveric lumbar spines (38-66 years old; 56 ± 10 years; Pfirrmann grades II-IV) were treated with 2.0 U/mL recombinant MMP-8 or control buffer (n = 6-12 CEP samples per group) for 18 h at 37°C and placed at the open sides of diffusion chambers. Bovine NP cells cultured inside the chambers were nourished by nutrients diffusing through the CEP tissues. After 72 hours in culture, depth-dependent NP cell viability and gene expression were measured using a live/dead assay and in situ hybridization. These cell viability and function outcomes were related to CEP transport properties and CEP biochemical composition, determined using fluorescence recovery after photobleaching (solute: 376 Da fluorescein) and Fourier transform infrared spectroscopy imaging. To test the effects of CEP matrix modification in whole discs, one CEP from each of six bovine discs was superficially treated with 0.75 U/mL collagenase or control buffer (n = 3 discs/group). Solute concentration inside the discs was measured after submerging the discs in fluorescein baths and applying dynamic compression (0.2 MPa for 6 h, then 0.4-0.8 MPa 0.5 Hz for 1.5 h). RESULTS Solute diffusivity in the CEPs varied nearly 4-fold (14.8 ± 5.1 μm2/s; range: 5.9–21.5 μm2/s), and chambers with the least permeable CEPs had up to 51% shorter viable distance from the CEP/nutrient interface. CEPs that permitted the least solute diffusion also attenuated the expression of aggrecan, type 2 collagen, and MMP-2 by the NP cells in the chambers. Doubling the chamber cell density shortened the viable distance; however, this effect depended on CEP transport properties. For CEPs with low diffusivity (<15 μm2/s), there was overlap in the relationships between viable distance and solute diffusivity. This suggests that in those cases, the main limiting factor was poor nutrient supply owing to low diffusive transport, not the increased nutrient demand caused by the higher number of cells. Quantitative spectroscopy revealed that low diffusivity CEPs had significantly higher amounts of collagen (Amide I peak, p = 0.0003) and aggrecan (carbohydrate peak, p = 0.002). Treating the CEPs with MMP-8 reduced the amounts of collagen (p < 0.05) and aggrecan (p < 0.05), and importantly, MMP-8-treated CEPs had 4-28% longer viable distance inside diffusion chambers (p = 0.01). Finally, bovine discs with enzyme-treated CEPs had up to 3.3-fold higher fluorescein concentration compared to site-matched locations in discs with buffer-treated CEPs (p = 0.003). CONCLUSIONS Cartilage endplate permeability has a significant effect on NP cell survival and function. Degeneration-related CEP matrix changes could hinder the success of biologic therapies that require increased nutrient supply. Treatment of the CEP with matrix-modifying enzymes can ameliorate these matrix changes and thereby improve disc nutrient supply for biologic therapy. FDA DEVICE/DRUG STATUS Human recombinant MMP-8, Clostridium collagenase (Not approved for this indication). Intradiscal biologic therapy (stem cell, gene, or growth factor) is a promising strategy for disc regeneration. However, these therapies require a rich nutrient supply, which may be limited by the permeability of the cartilage endplate (CEP). The goals of this research were to: 1) investigate how physiologic fluctuations in CEP permeability impact nutrient diffusion and nucleus pulposus (NP) cell survival and function; 2) identify compositional traits of the CEP matrix that coincide with reduced permeability; and 3) test if matrix modification of the CEP via enzymatic treatment with human MMP-8 can improve nutrient transport and NP cell survival. We hypothesized that deficits in CEP composition that hinder nutrient diffusion would associate with impaired NP cell survival and function, and that ameliorating those CEP compositional deficits via MMP-8 treatment would enhance nutrient diffusion and cell survival. This basic science study involved ex vivo analyses of cadaveric human CEP tissues, bovine NP cell culture, and biomechanical testing. Human CEP tissues harvested from six cadaveric lumbar spines (38-66 years old; 56 ± 10 years; Pfirrmann grades II-IV) were treated with 2.0 U/mL recombinant MMP-8 or control buffer (n = 6-12 CEP samples per group) for 18 h at 37°C and placed at the open sides of diffusion chambers. Bovine NP cells cultured inside the chambers were nourished by nutrients diffusing through the CEP tissues. After 72 hours in culture, depth-dependent NP cell viability and gene expression were measured using a live/dead assay and in situ hybridization. These cell viability and function outcomes were related to CEP transport properties and CEP biochemical composition, determined using fluorescence recovery after photobleaching (solute: 376 Da fluorescein) and Fourier transform infrared spectroscopy imaging. To test the effects of CEP matrix modification in whole discs, one CEP from each of six bovine discs was superficially treated with 0.75 U/mL collagenase or control buffer (n = 3 discs/group). Solute concentration inside the discs was measured after submerging the discs in fluorescein baths and applying dynamic compression (0.2 MPa for 6 h, then 0.4-0.8 MPa 0.5 Hz for 1.5 h). Solute diffusivity in the CEPs varied nearly 4-fold (14.8 ± 5.1 μm2/s; range: 5.9–21.5 μm2/s), and chambers with the least permeable CEPs had up to 51% shorter viable distance from the CEP/nutrient interface. CEPs that permitted the least solute diffusion also attenuated the expression of aggrecan, type 2 collagen, and MMP-2 by the NP cells in the chambers. Doubling the chamber cell density shortened the viable distance; however, this effect depended on CEP transport properties. For CEPs with low diffusivity (<15 μm2/s), there was overlap in the relationships between viable distance and solute diffusivity. This suggests that in those cases, the main limiting factor was poor nutrient supply owing to low diffusive transport, not the increased nutrient demand caused by the higher number of cells. Quantitative spectroscopy revealed that low diffusivity CEPs had significantly higher amounts of collagen (Amide I peak, p = 0.0003) and aggrecan (carbohydrate peak, p = 0.002). Treating the CEPs with MMP-8 reduced the amounts of collagen (p < 0.05) and aggrecan (p < 0.05), and importantly, MMP-8-treated CEPs had 4-28% longer viable distance inside diffusion chambers (p = 0.01). Finally, bovine discs with enzyme-treated CEPs had up to 3.3-fold higher fluorescein concentration compared to site-matched locations in discs with buffer-treated CEPs (p = 0.003). Cartilage endplate permeability has a significant effect on NP cell survival and function. Degeneration-related CEP matrix changes could hinder the success of biologic therapies that require increased nutrient supply. Treatment of the CEP with matrix-modifying enzymes can ameliorate these matrix changes and thereby improve disc nutrient supply for biologic therapy.

Poor solute transport through the cartilage endplate (CEP) impairs disc nutrition and could be a key factor that limits the success of intradiscal biologic therapies. Here we demonstrate that treating the CEP with matrix metalloproteinase-8 (MMP-8) reduces the matrix constituents that impede solute uptake and thereby improves nutrient diffusion. Human CEP tissues harvested from four fresh cadaveric lumbar spines (age range: 38-66 years old) were treated with MMP-8. Treatment caused a dose-dependent reduction in sGAG, localized reductions to the amount of collagen, and alterations to collagen structure. These matrix modifications corresponded with 16-24% increases in the uptake of a small solute (376 Da). Interestingly, the effects of MMP-8 treatment depended on the extent of non-enzymatic glycation: treated CEPs with high concentrations of advanced glycation end products (AGEs) exhibited the lowest uptake compared to treated CEPs with low concentrations of AGEs. Moreover, AGE concentrations were donor-specific, and the donor tissues with the highest AGE concentrations appeared to have lower uptake than would be expected based on the initial amounts of collagen and sGAG. Finally, increasing solute uptake in the CEP improved cell viability inside diffusion chambers, which supports the nutritional relevance of enhancing the transport properties of the CEP. Taken together, our results provide new insights and in vitro proof-of-concept for a treatment approach that could improve disc nutrition for biologic therapy: specifically, matrix reduction by MMP-8 can enhance solute uptake and nutrient diffusion through the CEP, and AGE concentration appears to be an important, patient-specific factor that influences the efficacy of this approach.

Collagen and hyaluronan are the most abundant components of the extracellular matrix (ECM) and their overexpression in tumors is linked to increased tumor growth and metastasis. These ECM components contribute to a protective tumor microenvironment by supporting a high interstitial fluid pressure and creating a tortuous setting for the convection and diffusion of chemotherapeutic small molecules, antibodies, and nanoparticles in the tumor interstitial space. This review focuses on the research efforts to deplete extracellular collagen with collagenases to normalize the tumor microenvironment. Although collagen synthesis inhibitors are in clinical development, the use of collagenases is contentious and clinically untested in cancer patients. Pretreatment of murine tumors with collagenases increased drug uptake and diffusion 2-10-fold. This modest improvement resulted in decreased tumor growth, but the benefits of collagenase treatment are confounded by risks of toxicity from collagen breakdown in healthy tissues. In this review, we evaluate the published in vitro and in vivo benefits and limitations of collagenase treatment to improve drug delivery.

Correction for Sterol-modified PEG lipids: alteration of the bilayer anchoring moiety has an unexpected effect on liposome circulation' by Aaron Dolor et al., Chem. Commun., 2018, 54, 11949-11952.

We synthesized and characterized two novel sterol-anchored polyethylene glycols (PEG) as potential alternatives to conventional phosphatidylethanolamine-PEGs. Liposomes containing the dicholesterol anchored PEG at 5 mole percent exhibit canonical PEGgylated-liposome behaviors including retention of encapsulated small molecules, low serum protein adsorption, and reduced cellular uptake yet they do not exhibit long circulation.

Correction for 'Sterol-modified PEG lipids: alteration of the bilayer anchoring moiety has an unexpected effect on liposome circulation' by Aaron Dolor et al., Chem. Commun., 2018, 54, 11949-11952.

Nitro-porphyrins are an important class of commercial dyes with a range of potential applications. The nitro group is known to dramatically affect the photophysics of the porphyrin, but there are few systematic investigations of the contributing factors. To address this deficiency, we present spectroscopic studies of a series of nitro-porphyrins, accompanied by density functional theory calculations to elucidate their structures. In particular, we explore how the positions of the substituents affect the energy levels and nuclear geometry. As expected, nitro groups on the meso-phenyl rings cause small changes to the orbital energies by induction, while those at the β-pyrrole positions more strongly conjugate into the aromatic system. In addition, however, we find evidence that β-pyrrole nitro groups distort the porphyrin, creating two non-planar conformations with distinct properties. This unexpected result helps explain the anomalous photophysics of nitro-porphyrins reported throughout the literature, including inhomogeneous line broadening and biexponential fluorescence decay. © 2017 Wiley Periodicals, Inc.

In the quest for better medicines, attention is increasingly turning to cell-based therapies. The rationale is that infused cells can provide a targeted therapy to precisely correct a complex disease phenotype. Between 1987 and 2010, autologous macrophages (MΦs) were used in clinical trials to treat a variety of human tumors; this approach provided a modest therapeutic benefit in some patients but no lasting remissions. These trials were initiated prior to an understanding of: the complexity of MΦ phenotypes, their ability to alter their phenotype in response to various cytokines and/or the environment, and the extent of survival of the re-infused MΦs. It is now known that while inflammatory MΦs can kill tumor cells, the tumor environment is able to reprogram MΦs into a tumorigenic phenotype; inducing blood vessel formation and contributing to a cancer cell growth-promoting milieu. We review how new information enables the development of large numbers of ex vivo generated MΦs, and how conditioning and gene engineering strategies are used to restrict the MΦ to an appropriate phenotype or to enable production of therapeutic proteins. We survey applications in which the MΦ is loaded with nanomedicines, such as liposomes ex vivo, so when the drug-loaded MΦs are infused into an animal, the drug is released at the disease site. Finally, we also review the current status of MΦ biodistribution and survival after transplantation into an animal. The combination of these recent advances opens the way for improved MΦ cell therapies.

We describe a new class of charge inverted zwitterionic sulfated lipids (AS) with a cationic quaternary amine anchored at the membrane interface and an anionic sulfate moiety extended into the aqueous phase. These lipids have exceptionally high transition temperatures and assemble into lipid aggregates when dispersed in aqueous solutions.

A simple, high yield, two-step synthesis yields a porphyrin dimer linked by a flexible dithiol tether that preferentially binds fullerene C-70 over C-60 in toluene solution. The complex forms stable aggregates when cast on glass.