Jeannine Coburn

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.

Bioinspired nanofibers support chondrogenesis for articular cartilage repair

Articular cartilage repair remains a significant and growing clinical challenge with the aging population. The native extracellular matrix (ECM) of articular cartilage is a 3D structure composed of proteinaceous fibers and a hydrogel ground substance that together provide the physical and biological cues to instruct cell behavior. Here we present fibrous scaffolds composed of poly(vinyl alcohol) and the biological cue chondroitin sulfate with fiber dimensions on the nanoscale for application to articular cartilage repair. The unique, low-density nature of the described nanofiber scaffolds allows for immediate cell infiltration for optimal tissue repair. The capacity for the scaffolds to facilitate cartilage-like tissue formation was evaluated in vitro. Compared with pellet cultures, the nanofiber scaffolds enhance chondrogenic differentiation of mesenchymal stems cells as indicated by increased ECM production and cartilage specific gene expression while also permitting cell proliferation. When implanted into rat osteochondral defects, acellular nanofiber scaffolds supported enhanced chondrogenesis marked by proteoglycan production minimally apparent in defects left empty. Furthermore, inclusion of chondroitin sulfate into the fibers enhanced cartilage-specific type II collagen synthesis in vitro and in vivo. By mimicking physical and biological cues of native ECM, the nanofiber scaffolds enhanced cartilaginous tissue formation, suggesting their potential utility for articular cartilage repair.

Photoactivated Composite Biomaterial for Soft Tissue Restoration in Rodents and in Humans

Photoactivated composite poly(ethylene glycol)–hyaluronic acid biomaterials demonstrate enhanced physicochemical properties for facial soft tissue reconstruction. Photogenic Polymers Can Fix the Flaws Some people just love the spotlight; apparently, some polymers do too. Here, Hillel et al. introduce a class of composite polymers that react favorably to light by crosslinking within minutes. These polymers, composed of synthetic poly(ethylene glycol) (PEG) and natural hyaluronic acid (HA), have been developed for reconstructing facial soft tissue. Deformities in craniofacial soft tissue are a clinical challenge because even small defects can have a major impact on a person’s social behavior and psychological well-being. Hillel and colleagues created a polymeric composite that can be injected into the damaged site, massaged into shape, and then crosslinked in situ with light. A transdermal light exposure method would allow clinicians to inject a liquid polymer, rather than surgically inserting already-polymerized material. First, the authors designed an array of light-emitting diodes to penetrate up to 4 mm of human skin (both light and dark) without any painful side effects. A 2-min exposure to light was enough to crosslink the PEG-HA material under the skin. Next, the polymer was tailored to closely match the elastic properties of native soft tissues, such as human fat. Various amounts of PEG and concentrations of HA were tested, with the authors arriving at an optimal combination of 100 mg PEG and 24 mg/ml HA. When polymerized subcutaneously in rats, the PEG-HA implants were able to maintain near their original volume for up to 491 days, whereas control HA injections were completely resorbed. Notably, these HA-based materials were partially reversible with the addition of the enzyme hyaluronidase. To translate this material to the clinic, Hillel et al. then tested the PEG-HA composites in humans. The polymer was injected into the intradermal space in the abdomen of three patients scheduled to undergo abdominoplasty surgery. Similar to the rodent studies, the PEG-HA material persisted for 12 weeks, whereas the control HA injections lost their shape. An inflammatory response was observed surrounding the injections. It is clear that this new photo-friendly polymer and transdermal crosslinking method will be clinically useful for soft tissue reconstruction—perhaps even encouraging more people to put their best faces forward in the spotlight. Soft tissue reconstruction often requires multiple surgical procedures that can result in scars and disfiguration. Facial soft tissue reconstruction represents a clinical challenge because even subtle deformities can severely affect an individual’s social and psychological function. We therefore developed a biosynthetic soft tissue replacement composed of poly(ethylene glycol) (PEG) and hyaluronic acid (HA) that can be injected and photocrosslinked in situ with transdermal light exposure. Modulating the ratio of synthetic to biological polymer allowed us to tune implant elasticity and volume persistence. In a small-animal model, implanted photocrosslinked PEG-HA showed a dose-dependent relationship between increasing PEG concentration and enhanced implant volume persistence. In direct comparison with commercial HA injections, the PEG-HA implants maintained significantly greater average volumes and heights. Reversibility of the implant volume was achieved with hyaluronidase injection. Pilot clinical testing in human patients confirmed the feasibility of the transdermal photocrosslinking approach for implantation in abdomen soft tissue, although an inflammatory response was observed surrounding some of the materials.

Laser-based three-dimensional multiscale micropatterning of biocompatible hydrogels for customized tissue engineering scaffolds

Significance In this paper we present results on 3D, multiscale laser machining of soft, transparent biomaterials suited for cellular growth and/or implantation. We use an ultrafast laser to generate high-resolution, 3D structures within the bulk of a transparent soft-biomaterial formulation that can support cell growth and allow cells to penetrate deep within the material. The structure is created by multiphoton absorption which, thanks to the clarity of the silk gels, is possible nearly 1 cm below the surface of the material. This depth represents an ∼10× improvement over other materials. The ability to create micrometer-scale voids over such a large volume has promising applications in the biomedical field and its efficacy was demonstrated both in vitro and in vivo. Light-induced material phase transitions enable the formation of shapes and patterns from the nano- to the macroscale. From lithographic techniques that enable high-density silicon circuit integration, to laser cutting and welding, light–matter interactions are pervasive in everyday materials fabrication and transformation. These noncontact patterning techniques are ideally suited to reshape soft materials of biological relevance. We present here the use of relatively low-energy (< 2 nJ) ultrafast laser pulses to generate 2D and 3D multiscale patterns in soft silk protein hydrogels without exogenous or chemical cross-linkers. We find that high-resolution features can be generated within bulk hydrogels through nearly 1 cm of material, which is 1.5 orders of magnitude deeper than other biocompatible materials. Examples illustrating the materials, results, and the performance of the machined geometries in vitro and in vivo are presented to demonstrate the versatility of the approach.

Size of the embryoid body influences chondrogenesis of mouse embryonic stem cells.

For applications in tissue engineering and regenerative medicine, embryonic stem cells (ESCs) are commonly pre‐differentiated in the form of embryoid bodies (EBs). The uncontrolled cell differentiation in EBs results in a highly heterogeneous cell population, an unfavourable condition for therapeutic development. The purpose of this study was to determine an optimal size of EBs for chondrogenic differentiation. EBs were produced in suspension culture with mouse ESCs (ES‐D3 GL). The 5‐day‐old EBs were sorted under a microscope by diameter: small EBs (S‐EBs, < 100 µm), medium EBs (M‐EBs, 100–150 µm) and large EBs (L‐EBs, > 150 µm). The three sizes of EBs were cultured separately for 3 weeks in chondrogenic medium. Type II collagen and aggrecan gene expression was significantly upregulated in the S‐EBs, when compared with the M‐EBs and L‐EBs (p < 0.05 and p < 0.001, respectively). Proteoglycans produced by the cells derived from S‐EBs were > 50% of the other two groups. In addition, both Oct4 and Sox2 were expressed more in S‐EBs than in M‐EBs and L‐EBs. Type X collagen expression was relatively increased in L‐EBs. Slight shifts toward haematopoietic and endothelial differentiation were seen in the L‐ and M‐EBs. In summary, the size of EBs has implications on ESC differentiation. Cells derived from S‐EBs have a greater chondrogenic potential than those from M‐EBs and L‐EBs. The size of EBs can be a parameter utilized to optimize ESC differentiation for tissue engineering. Copyright

Impact of silk biomaterial structure on proteolysis.

The goal of this study was to determine the impact of silk biomaterial structure (e.g. solution, hydrogel, film) on proteolytic susceptibility. In vitro enzymatic degradation of silk fibroin hydrogels and films was studied using a variety of proteases, including proteinase K, protease XIV, α-chymotrypsin, collagenase, matrix metalloproteinase-1 (MMP-1) and MMP-2. Hydrogels were used to assess bulk degradation while films were used to assess surface degradation. Weight loss, secondary structure determined by Fourier transform infrared spectroscopy and degradation products analyzed via sodium dodecyl sulfate-polyacrylamide gel electrophoresis were used to evaluate degradation over 5 days. Silk films were significantly degraded by proteinase K, while silk hydrogels were degraded more extensively by protease XIV and proteinase K. Collagenase preferentially degraded the β-sheet content in hydrogels while protease XIV and α-chymotrypsin degraded the amorphous structures. MMP-1 and MMP-2 degraded silk fibroin in solution, resulting in a decrease in peptide fragment sizes over time. The link between primary sequence mapping with protease susceptibility provides insight into the role of secondary structure in impacting proteolytic access by comparing solution vs. solid state proteolytic susceptibility.

Tissue engineering strategies to study cartilage development, degeneration and regeneration ☆

Cartilage tissue engineering has primarily focused on the generation of grafts to repair cartilage defects due to traumatic injury and disease. However engineered cartilage tissues have also a strong scientific value as advanced 3D culture models. Here we first describe key aspects of embryonic chondrogenesis and possible cell sources/culture systems for in vitro cartilage generation. We then review how a tissue engineering approach has been and could be further exploited to investigate different aspects of cartilage development and degeneration. The generated knowledge is expected to inform new cartilage regeneration strategies, beyond a classical tissue engineering paradigm.

Surgery combined with controlled-release doxorubicin silk films as a treatment strategy in an orthotopic neuroblastoma mouse model

Background:Neuroblastoma tumour resection goal is maximal tumour removal. We hypothesise that combining surgery with sustained, local doxorubicin application can control tumour growth.Methods:We injected human neuroblastoma cells into immunocompromised mouse adrenal gland. When KELLY cell-induced tumour volume was >300 mm3, 80–90% of tumour was resected and treated as follows: instantaneous-release silk film with 100 μg doxorubicin (100IR), controlled-release film with 200 μg (200CR) over residual tumour bed; and 100 and 200 μg intravenous doxorubicin (100IV and 200IV). Tumour volume was measured and histology analysed.Results:Orthotopic tumours formed with KELLY, SK-N-AS, IMR-32, SH-SY5Y cells. Tumours reached 1800±180 mm3 after 28 days, 2200±290 mm3 after 35 days, 1280±260 mm3 after 63 days, and 1700±360 mm3 after 84 days, respectively. At 3 days post KELLY tumour resection, tumour volumes were similar across all groups (P=0.6210). Tumour growth rate was similar in untreated vs control film, 100IV vs 100IR, and 100IV vs 200IV. There was significant difference in 100IR vs 200CR (P=0.0004) and 200IV vs 200CR (P=0.0003). Tumour growth with all doxorubicin groups was slower than that of control (P: <0.0001–0.0069). At the interface of the 200CR film and tumour, there was cellular necrosis, surrounded by apoptotic cells before reaching viable tumour cells.Conclusions:Combining surgical resection and sustained local doxorubicin treatment is effective in tumour control. Administering doxorubicin in a local, controlled manner is superior to giving an equivalent intravenous dose in tumour control.

Focal therapy of neuroblastoma using silk films to deliver kinase and chemotherapeutic agents in vivo

Current methods for treatment of high-risk neuroblastoma patients include surgical intervention, in addition to systemic chemotherapy. However, only limited therapeutic tools are available to pediatric surgeons involved in neuroblastoma care, so the development of intraoperative treatment modalities is highly desirable. This study presents a silk film library generated for focal therapy of neuroblastoma; these films were loaded with either the chemotherapeutic agent doxorubicin or the targeted drug crizotinib. Drug release kinetics from the silk films were fine-tuned by changing the amount and physical crosslinking of silk; doxorubicin loaded films were further refined by applying a gold nanocoating. Doxorubicin-loaded, physically crosslinked silk films showed the best in vitro activity and superior in vivo activity in orthotopic neuroblastoma studies when compared to the doxorubicin-equivalent dose administered intravenously. Silk films were also suitable for delivery of the targeted drug crizotinib, as crizotinib-loaded silk films showed an extended release profile and an improved response both in vitro and in vivo when compared to freely diffusible crizotinib. These findings, when combined with prior in vivo data on silk, support a viable future for silk-based anticancer drug delivery systems.

Photocrosslinking of Silk Fibroin Using Riboflavin for Ocular Prostheses

A novel method to photocrosslink silk fibroin protein is reported, using riboflavin (vitamin B2) as a photoinitiator and the mechanism of crosslinking is determined. Exposure of riboflavin-doped liquid silk solution to light results in the formation of a transparent, elastic hydrogel. Several applications for this new material are investigated including corneal reshaping to restore visual acuity and photolithography.