Kartogenin Enhances Collagen Organization and Mechanical Strength of the Repaired Enthesis in a Murine Model of Rotator Cuff Repair.

PURPOSE: To investigate the use of kartogenin (KGN) in augmenting healing of the repaired enthesis after rotator cuff repair in a murine model. METHODS: Seventy-two C57BL/6 wild-type mice underwent unilateral detachment and transosseous repair of the supraspinatus tendon augmented with either fibrin sealant (control group; n = 36) or fibrin sealant containing 100 µmol/L of KGN (experimental group; n = 36) applied at the repair site. Postoperatively, mice were allowed free cage activity without immobilization. Mice were humanely killed at 2 and 4 weeks postoperatively. Repair site integrity was evaluated histologically through fibrocartilage formation and collagen fiber organization and biomechanically through load-to-failure testing of the supraspinatus tendon-bone construct. RESULTS: At 2 weeks, no differences were noted in percent area of fibrocartilage, collagen organization, or ultimate strength between groups. At 4 weeks, superior collagen fiber organization (based on collagen birefringence [17.3 ± 2.0 vs 7.0 ± 6.5 integrated density/µm2; P < .01]) and higher ultimate failure loads (3.5 ± 0.6 N vs 2.3 ± 1.1 N; P = .04) were seen in the KGN group. The percent area of fibrocartilage (13.2 ± 8.4% vs 4.4 ± 5.4%; P = .04) was higher in the control group compared with the KGN group. CONCLUSIONS: Rotator cuff repair augmentation with KGN improved the collagen fiber organization and biomechanical strength of the tendon-bone interface at 4 weeks in a murine model. CLINICAL RELEVANCE: These findings have implications for improving the structural integrity of the repaired enthesis and potentially reducing the retear rate after rotator cuff repair, which can ultimately lead to improvements in clinical outcomes.

Total Mastectomy or Breast Conservation Therapy? How Radiation Oncologist Accessibility Determines Treatment Choice and Quality: A SEER Data-base Analysis.

Mastectomy and breast conservation therapy (BCT) are equivalent in survival for treatment of early stage breast cancer. This study evaluated the impact of radiation oncologist accessibility on choice of breast conserving surgery (BCS) versus mastectomy, and the appropriate receipt of radiotherapy after BCS. In the National Cancer Institute Survival, Epidemiology, and End Results data base, the authors selected breast cancer cases from 2004 to 2008 with the following criteria: T2N1M0 or less, lobular or ductal histology, and treatment with simple or partial mastectomy. We combined the Health Resources and Services Administration Area Resource File to define average radiation oncologist density (ROD) by county over the same time period. We evaluated tumor characteristics, demographic information, and ROD with respect to BCS rates and receipt of radiation therapy after BCS in univariable and multivariable analyses. In 118,773 cases analyzed, mastectomy was performed 33.2% of the time relative to BCS. After adjustment for demographic and tumor variables, the odds of having BCS versus mastectomy were directly associated with ROD (multiplicative change in odds for a single unit increase in ROD [95% CI] = 1.02 [1.01-1.03]; p < 0.001). Adjuvant radiation therapy was not administered in 28.2% of BCS cases. When adjusting for demographic and tumor variables, the odds of having BCS without adjuvant radiation were inversely associated with ROD (0.95 [0.94-0.97]; p < 0.001). We observed a direct relationship between ROD and BCS rates independent of demographic and tumor variables, and an inverse trend for omission of radiotherapy after BCS. Access to radiation oncologists may represent an important factor in surgical choice and receiving appropriate BCT in early stage breast cancer.

Light Absorptive Properties of Articular Cartilage, ECM Molecules, Synovial Fluid, and Photoinitiators as Potential Barriers to Light-Initiated Polymer Scaffolding Procedures.

OBJECTIVE: Many in vivo procedures to repair chondral defects use ultraviolet (UV)-photoinitiated in situ polymerization within the cartilage matrix. Chemical species that absorb UV light might reduce the effectiveness of these procedures by acting as light absorption barriers. This study evaluated whether any of the individual native biochemical components in cartilage and synovial fluid interfered with the absorption of light by common scaffolding photosensitizers. MATERIALS: UV-visible spectroscopy was performed on each major component of cartilage in solution, on bovine synovial fluid, and on four photosensitizers, riboflavin, Irgacure 2959, quinine, and riboflavin-5'-phosphate. Molar extinction and absorption coefficients were calculated at wavelengths of maximum absorbance and 365 nm. Intact articular cartilage was also examined. RESULTS: The individual major biochemical components of cartilage, Irgacure 2959, and quinine did not exhibit a significant absorption at 365 nm. Riboflavin and riboflavin-5'-phosphate were more effectual light absorbers at 365 nm, compared with the individual native species. Intact cartilage absorbed a significantly greater amount of UV light in comparison with the native species. CONCLUSION: Our results indicate that none of the individual native species in cartilage will interfere with the absorption of UV light at 365 nm by these commonly used photoinitiators. Intact cartilage slices exhibited significant light absorption at 365 nm, while also having distinct absorbance peaks at wavelengths less than 300 nm. Determining the UV absorptive properties of the biomolecules native to articular cartilage and synovial fluid will aid in optimizing scaffolding procedures to ensure sufficient scaffold polymerization at a minimum UV intensity.

Locally Applied Simvastatin as an Adjunct to Promote Spinal Fusion in Rats.

STUDY DESIGN: Basic Science. OBJECTIVE: To determine if locally delivered simvastatin can enhance bone formation in a rat spinal fusion model. SUMMARY OF BACKGROUND DATA: The bone-anabolic properties of statins in fracture healing are well established, however, few studies have evaluated the impact of locally delivered statins in spinal fusion. METHODS: We formulated poly(lactic-co-glycolic acid) (PLGA) nanoparticles by adapting previously published techniques. Two types of nanoparticles were created: simvastatin nanoparticles (SimNP) and nanoparticles without simvastatin (BlankNP). Drug elution from SimNP was characterized. Osteoblastic differentiation was analyzed using MC3T3-E1 cells cultured in differentiation medium containing SimNP or BlankNP. Forty male 12 week old outbred Wistar rats underwent uninstrumented posterolateral fusion using iliac crest bone graft and BlankNP, SimNP or simvastatin drug. X-rays to assess bone formation were obtained at 4 weeks and 9 weeks post-operatively. Spines were explanted at 9 weeks for micro-CT analysis, and a blinded manual assessment of fusion (MAF). RESULTS: SimNP achieved a release efficiency of 74.1% with ∼50% release occurring in the first day. Simvastatin and SimNP treated cells showed significantly greater expression of osteopontin (OPN) and osteocalcin (OCN). On micro-CT analysis, SimNP animals had higher bone volume and percent bone volume (bone volume/total volume) than control animals. SimNP rats had higher X-ray scores at 4 weeks (p=0.010) and 9 weeks (p<0.001) relative to BlankNP. MAF showed that SimNP had a higher fusion rate than BlankNP (42.9% vs. 0%, p=0.006). CONCLUSION: We were able to validate that sustained release of simvastatin via a PLGA nanoparticle. SimNP was able to induce an increase in mineralization as well as an increase in markers of bone formation. X-ray analysis, micro-CT quantification, and MAF assessment of SimNP treated rats showed significantly greater bone formation and fusion mass strength relative to vehicle treated animals. Simvastatin may be a safe, cost-effective bone anabolic agent for use in spinal fusion. LEVEL OF EVIDENCE: N/A.

Self-assembled monolayers of phosphonates promote primary chondrocyte adhesion to silicon dioxide and polyvinyl alcohol materials.

The optimal solution for articular cartilage repair has not yet been identified, in part because of the challenges in achieving integration with the host. Coatings have the potential to transform the adhesive features of surfaces, but their application to cartilage repair has been limited. Self-assembled monolayer of phosphonates (SAMPs) have been demonstrated to increase the adhesion of various immortalized cell types to metal and polymer surfaces, but their effect on primary chondrocyte adhesion has not been studied. The objective of this study was to investigate the response of primary chondrocytes to SAMP coatings. We hypothesized a SAMP terminated with an α,ω-bisphosphonic acid, in particular butane-1,4-diphosphonic acid, would increase the number of adherent primary chondrocytes to polyvinyl alcohol (PVA). To test our hypothesis, we first established our ability to successfully modify silicon dioxide (SiO2) surfaces to enable chondrocytes to attach to the surface, without substantial changes in gene expression. Secondly, we applied identical chemistry to PVA, and quantified chondrocyte adhesion. SAMP modification to SiO2 increased chondrocyte adhesion by ×3 after 4 hr and ×4.5 after 24 hr. PVA modification with SAMPs increased chondrocyte adhesion by at least ×31 after 4 and 24 hours. Changes in cell morphology indicated that SAMP modification led to improved chondrocyte adhesion and spreading, without changes in gene expression. In summary, we modified SiO2 and PVA with SAMPs and observed an increase in the number of adherent primary bovine chondrocytes at 4 and 24 hr post-seeding. Mechanisms of chondrocyte interaction with SAMP-modified surfaces require further investigation.

Photocrosslinked tyramine-substituted hyaluronate hydrogels with tunable mechanical properties improve immediate tissue-hydrogel interfacial strength in articular cartilage.

Articular cartilage lacks the ability to self-repair and a permanent solution for cartilage repair remains elusive. Hydrogel implantation is a promising technique for cartilage repair; however for the technique to be successful hydrogels must interface with the surrounding tissue. The objective of this study was to investigate the tunability of mechanical properties in a hydrogel system using a phenol-substituted polymer, tyramine-substituted hyaluronate (TA-HA), and to determine if the hydrogels could form an interface with cartilage. We hypothesized that tyramine moieties on hyaluronate could crosslink to aromatic amino acids in the cartilage extracellular matrix. Ultraviolet (UV) light and a riboflavin photosensitizer were used to create a hydrogel by tyramine self-crosslinking. The gel mechanical properties were tuned by varying riboflavin concentration, TA-HA concentration, and UV exposure time. Hydrogels formed with a minimum of 2.5 min of UV exposure. The compressive modulus varied from 5 to 16 kPa. Fluorescence spectroscopy analysis found differences in dityramine content. Cyanine-3 labelled tyramide reactivity at the surface of cartilage was dependent on the presence of riboflavin and UV exposure time. Hydrogels fabricated within articular cartilage defects had increasing peak interfacial shear stress at the cartilage-hydrogel interface with increasing UV exposure time, reaching a maximum shear stress 3.5× greater than a press-fit control. Our results found that phenol-substituted polymer/riboflavin systems can be used to fabricate hydrogels with tunable mechanical properties and can interface with the surface tissue, such as articular cartilage.

Perforation Does Not Compromise Patterned Two-Dimensional Substrates for Cell Attachment and Aligned Spreading.

Polymeric sheets were perforated by laser ablation and were uncompromised by a debris field when first treated with a thin layer of photoresist. Polymer sheets perforated with holes comprising 5, 10, and 20% of the nominal surface area were then patterned in stripes by photolithography, which was followed by synthesis in exposed regions of a cell-attractive zirconium oxide-1,4-butanediphosphonic acid interface. Microscopic and scanning electron microscopy analyses following removal of unexposed photoresist show well-aligned stripes for all levels of these perforations. NIH 3T3 fibroblasts plated on each of these perforated surfaces attached to the interface and spread in alignment with pattern fidelity in every case that is as high as that measured on a nonperforated, patterned substrate.

Resurfacing damaged articular cartilage to restore compressive properties.

Surface damage to articular cartilage is recognized as the initial underlying process causing the loss of mechanical function in early-stage osteoarthritis. In this study, we developed structure-modifying treatments to potentially prevent, stabilize or reverse the loss in mechanical function. Various polymers (chondroitin sulfate, carboxymethylcellulose, sodium hyaluronate) and photoinitiators (riboflavin, irgacure 2959) were applied to the surface of collagenase-degraded cartilage and crosslinked in situ using UV light irradiation. While matrix permeability and deformation significantly increased following collagenase-induced degradation of the superficial zone, resurfacing using tyramine-substituted sodium hyaluronate and riboflavin decreased both values to a level comparable to that of intact cartilage. Repetitive loading of resurfaced cartilage showed minimal variation in the mechanical response over a 7 day period. Cartilage resurfaced using a low concentration of riboflavin had viable cells in all zones while a higher concentration resulted in a thin layer of cell death in the uppermost superficial zone. Our approach to repair surface damage initiates a new therapeutic advance in the treatment of injured articular cartilage with potential benefits that include enhanced mechanical properties, reduced susceptibility to enzymatic degradation and reduced adhesion of macrophages.

A Simple Nanoscale Interface Directs Alignment of a Confluent Cell Layer on Oxide and Polymer Surfaces.

Templating of cell spreading and proliferation is described that yields confluent layers of cells aligned across an entire two-dimensional surface. The template is a reactive, two-component interface that is synthesized in three steps in nanometer thick, micron-scaled patterns on silicon and on several biomaterial polymers. In this method, a volatile zirconium alkoxide complex is first deposited at reduced pressure onto a surface pattern that is prepared by photolithography; the substrate is then heated to thermolyze the organic ligands to form surface-bound zirconium oxide patterns. The thickness of this oxide layer ranges from 10 to 70 nanometers, which is controlled by alkoxide complex deposition time. The oxide layer is treated with 1,4-butanediphosphonic acid to give a monolayer pattern whose composition and spatial conformity to the photolithographic mask are determined spectroscopically. NIH 3T3 fibroblasts and human bone marrow-derived mesenchymal stem cells attach and spread in alignment with the pattern without constraint by physical means or by arrays of cytophilic and cytophobic molecules. Cell alignment with the pattern is maintained as cells grow to form a confluent monolayer across the entire substrate surface.

Serum vitamin D levels among patients in a clinical oncology practice compared to primary care patients in the same community: a case-control study.

Objectives Low serum vitamin D levels have been associated with risk for certain malignancies, but studies have not directly analysed levels between community oncology and primary care practices. The purpose of this study was to compare serum vitamin D levels in patients at a community oncology practice with non-cancer patients at a primary care practice. Design Retrospective case-control study. 25-Hydroxyvitamin D levels were ordered for screening in both cancer and non-cancer patients. Levels were compared in univariate and multivariate analyses adjusted for age, body mass index and season of blood draw. Setting A community-based radiation oncology centre and a community-based primary care practice: both located in Northeastern Pennsylvania, USA. Participants 170 newly diagnosed cancer patients referred for initial consultation at the community oncology centre from 21 November 2008 to 18 May 2010, and 170 non-cancer patients of the primary care practice who underwent screening for hypovitaminosis D for the first time from 1 January 2009 to 31 December 2009. Primary and secondary outcome measures The primary outcome measure was mean serum vitamin D level, and the secondary outcome measures were frequencies of patients with vitamin D levels <20 ng/ml and levels <30 ng/ml. Results The oncology patients had a significantly lower mean serum vitamin D level (24.9 ng/ml) relative to a cohort of non-cancer primary care patients (30.6 ng/ml, p<0.001) from the same geographical region. The relationship retained significance after adjustment for age, body mass index and season of blood draw in multivariate analysis (p=0.001). Levels <20 and <30 ng/ml were more frequent in the oncology patients (OR (95% CI)=2.59 (1.44 to 4.67) and 2.04 (1.20 to 3.46), respectively) in multivariate analysis. Conclusions Cancer patients were found to have low vitamin D levels relative to a similar cohort of non-cancer primary care patients from the same geographical region.

A nanoscale interface improves attachment of cast polymers to glass.

A novel interface was prepared on glass slides that stabilizes several cast polymers against delamination under conditions necessary for the study of cell surface interactions. This interface was synthesized by deposition of zirconium tetra(tert-butoxide) from the vapor phase onto the glass followed by mild thermolysis, which gives a surface-bound zirconium oxide coating. This oxide coating improved attachment of polymer coatings cast from formic acid or methylene chloride. Nylon, polyurethane, and polyhydroxybutyrate/polyhydroxyvalerate coatings were stable against delamination from the oxide-coated glass following sonication in ethanol for more than 30 min or immersion in water at pH 8 for at least 48 h.

Vitamin enhanced waters and polyphenol rich beverages analyzed for antioxidant capacity and antioxidants/calorie.

The purpose of this study was to analyze polyphenol rich beverages (vitamin enhanced waters (VEWs), fruit juices and berry juices) to determine free polyphenol concentrations and free polyphenols per Calorie based on a serving size. The Folin-Ciocalteu reagent was used in a colorimetric assay based on a catechin standard. Fruit and berry juices contained, on average, more than eight-times the concentration of free polyphenols when compared to VEWs. When Calories per serving were taken into consideration, fruit and berry juices contained more than twice the free polyphenols per Calorie.