Sara Fermanian

Human Cartilage Repair with a Photoreactive Adhesive-Hydrogel Composite

A photoactive hydrogel is used in combination with microfracture to heal cartilage defects in patients. Let There Be Light Light has long been a favorite tool in medicine, finding utility in everything from skin conditions to depression to imaging. Now, Sharma and colleagues have shown that light can be used for biomaterials. Shining light on a hydrogel mixture causes it to polymerize within a defect, thus promoting tissue growth and repairing cartilage in patients. The biomaterial was designed to fill irregular wounds, such as articular cartilage defects. A biological adhesive was applied to the defect, followed by filling with a poly(ethylene glycol) (PEG)–based hydrogel solution. Then, light was applied to polymerize the material to form a solid implant. The hydrogel-adhesive was tested in a large-animal model to see how it worked in combination with the standard procedure for cartilage repair, called microfracture. The surgeons noted that the animals that received the biomaterial along with microfracture had a greater defect fill that was stronger and had more heterogeneous components (cells, proteins, etc.). The authors then moved to testing in people. Fifteen patients with symptomatic cartilage defects were treated with the adhesive-hydrogel after microfracture, whereas three patients were treated with microfracture only. No major adverse events were noted in 6 months after surgery. Similar to the animal studies, the photoactive biomaterial allowed for a greater filling of repair tissue in the defect compared with the control group, with material properties similar to adjacent, healthy cartilage. In addition, hydrogel-treated patients reported a decrease in overall pain severity and frequency over time. Although further clinical testing is needed to compare long-term outcomes in more patients, this light-mediated biomaterial therapy promises to be a versatile and safe way to enhance cartilage repair. Surgical options for cartilage resurfacing may be significantly improved by advances and application of biomaterials that direct tissue repair. A poly(ethylene glycol) diacrylate (PEGDA) hydrogel was designed to support cartilage matrix production, with easy surgical application. A model in vitro system demonstrated deposition of cartilage-specific extracellular matrix in the hydrogel biomaterial and stimulation of adjacent cartilage tissue development by mesenchymal stem cells. For translation to the joint environment, a chondroitin sulfate adhesive was applied to covalently bond and adhere the hydrogel to cartilage and bone tissue in articular defects. After preclinical testing in a caprine model, a pilot clinical study was initiated where the biomaterials system was combined with standard microfracture surgery in 15 patients with focal cartilage defects on the medial femoral condyle. Control patients were treated with microfracture alone. Magnetic resonance imaging showed that treated patients achieved significantly higher levels of tissue fill compared to controls. Magnetic resonance spin-spin relaxation times (T2) showed decreasing water content and increased tissue organization over time. Treated patients had less pain compared with controls, whereas knee function [International Knee Documentation Committee (IKDC)] scores increased to similar levels between the groups over the 6 months evaluated. No major adverse events were observed over the study period. With further clinical testing, this practical biomaterials strategy has the potential to improve the treatment of articular cartilage defects.

Metabolic changes in mesenchymal stem cells in osteogenic medium measured by autofluorescence spectroscopy

The purpose of this study was to measure metabolic changes in mesenchymal stem cells (MSCs) placed in osteogenic medium by autofluorescence spectroscopy. MSCs were plated in stem cell‐supporting or osteogenic medium and imaged. Shift from the basic growth environment to the inductive osteogenic environment was confirmed by reverse transcription‐polymerase chain reaction. Reduced pyridine nucleotides were detected by exciting near 366 nm and measuring fluorescence at 450 nm, and oxidized flavoproteins were detected by exciting at 460 nm and measuring fluorescence at 540 nm. The ratio of these fluorescence measurements, reduction‐oxidation (redox) fluorometry, is a noninvasive measure of the cellular metabolic state. The detected pyridine nucleotide to flavoprotein ratio decreased upon transitioning from the stem cell to the differentiated state, as well as with increasing cell density and cell‐cell contact. MSC metabolism increased upon placement in differentiating medium and with increasing cell density and contact. Redox fluorometry is a feasible, noninvasive technique for distinguishing MSCs from further differentiated cells.

Keratocyte behavior in three-dimensional photopolymerizable poly(ethylene glycol) hydrogels

The goal of this study was to evaluate three-dimensional (3-D) poly(ethylene glycol) (PEG) hydrogels as a culture system for studying corneal keratocytes. Bovine keratocytes were subcultured in DMEM/F-12 containing 10% fetal bovine serum (FBS) through passage 5. Primary keratocytes (P0) and corneal fibroblasts from passages 1 (P1) and 3 (P3) were photoencapsulated at various cell concentrations in PEG hydrogels via brief exposure to light. Additional hydrogels contained adhesive YRGDS and nonadhesive YRDGS peptides. Hydrogel constructs were cultured in DMEM/F-12 with 10% FBS for 2 and 4 weeks. Cell viability was assessed by DNA quantification and vital staining. Biglycan, type I collagen, type III collagen, keratocan and lumican expression were determined by reverse transcriptase-polymerase chain reaction. Deposition of type I collagen, type III collagen and keratan sulfate (KS)-containing matrix components was visualized using confocal microscopy. Keratocytes in a monolayer lost their stellate morphology and keratocan expression, displayed elongated cell bodies, and up-regulated biglycan, type I collagen and type III collagen characteristic of corneal fibroblasts. Encapsulated keratocytes remained viable for 4 weeks with spherical morphologies. Hydrogels supported production of KS, type I collagen and type III collagen matrix components. PEG-based hydrogels can support keratocyte viability and matrix production. 3-D hydrogel culture can stabilize but not restore the keratocyte phenotype. This novel application of PEG hydrogels has potential use in the study of corneal keratocytes in a 3-D environment.