Thermal stabilization of diverse biologics using reversible hydrogels.

Improving the thermal stability of biologics, including vaccines, is critical to reduce the economic costs and health risks associated with the cold chain. Here, we designed a versatile, safe, and easy-to-use reversible PEG-based hydrogel platform formed via dynamic covalent boronic ester cross-linking for the encapsulation, stabilization, and on-demand release of biologics. Using these reversible hydrogels, we thermally stabilized a wide range of biologics up to 65°C, including model enzymes, heat-sensitive clinical diagnostic enzymes (DNA gyrase and topoisomerase I), protein-based vaccines (H5N1 hemagglutinin), and whole viruses (adenovirus type 5). Our data support a generalized protection mechanism for the thermal stabilization of diverse biologics using direct encapsulation in reversible hydrogels. Furthermore, preliminary toxicology data suggest that the components of our hydrogel are safe for in vivo use. Our reversible hydrogel platform offers a simple material solution to mitigate the costs and risks associated with reliance on a continuous cold chain for biologic transport and storage.

Stress Relaxation and Composition of Hydrazone-Crosslinked Hybrid Biopolymer-Synthetic Hydrogels Determine Spreading and Secretory Properties of MSCs.

The extracellular matrix plays a critical role in mechanosensing and thereby influences the secretory properties of bone-marrow-derived mesenchymal stem/stromal cells (MSCs). As a result, interest has grown in the development of biomaterials with tunable properties for the expansion and delivery of MSCs that are used in cell-based therapies. Herein, stress-relaxing hydrogels are synthesized as hybrid networks containing both biopolymer and synthetic macromer components. Hyaluronic acid is functionalized with either aldehyde or hydrazide groups to form covalent adaptable hydrazone networks, which are stabilized by poly(ethylene glycol) functionalized with bicyclononyne and heterobifunctional small molecule crosslinkers containing azide and benzaldehyde moieties. Tuning the composition of these gels allows for controlled variation in the characteristic timescale for stress relaxation and the amount of stress relaxed. Over this compositional space, MSCs are observed to spread in formulations with higher degrees of adaptability, with aspect ratios of 1.60 ± 0.18, and YAP nuclear:cytoplasm ratios of 6.5 ± 1.3. Finally, a maximum MSC pericellular protein thickness of 1.45 ± 0.38 µm occurred in highly stress-relaxing gels, compared to 1.05 ± 0.25 µm in non-adaptable controls. Collectively, this study contributes a new understanding of the role of compositionally defined stress relaxation on MSCs mechanosensing and secretion.

The utility of magnetic resonance imaging results in physician decision-making before initial lumbar spinal injection.

BACKGROUND CONTEXT: The need for advanced imaging before spinal intervention is an area of ongoing debate. Many studies have demonstrated the accuracy of magnetic resonance imaging (MRI) results in evaluating structural pathology in the lumbar spine, but few have addressed how frequently MRI findings change clinical management. A randomized controlled trial showed that viewing MRI results did not impact outcomes in patients with radiculopathy undergoing epidural steroid injection (ESI). The results suggested ESIs that correlated with both imaging and clinical findings experienced slightly more benefit than the blinded cohort, although statistically insignificantly. PURPOSE: Three related studies were conducted to (1) increase understanding of the opinions of interventional spine physicians regarding the utility of viewing imaging before injection and (2) evaluate the impact of viewing MRI results on injection planning (retrospective and prospective analyses). STUDY DESIGN: Survey, prospective, and retrospective analysis. PATIENT SAMPLE: Patients presenting to a university-based spine center for initial evaluation of back or leg pain who were candidates for spinal intervention. OUTCOME MEASURES: Self-reported measures from a clinical practice questionnaire distributed to interventional spine physicians to determine rates and rationale for utilization of MRI before spine injection, physiologic measures including MRI results, functional measures including physician decision-making regarding type and location of injection performed. METHODS: This study was funded by the University of Colorado Health and Welfare Trust. A survey was sent to interventional spine physicians to assess their utilization of MRI results before spine procedures. A retrospective analysis of patients who were candidates for ESI was conducted to evaluate how initial injection plan compared with the postviewing of MRI results on injection performed. In a prospective analysis, injection plans pre- and post-MRI were compared among patients presenting for initial evaluation of low back or leg pain. RESULTS: Survey responses showed that specialists order MRI studies to correlate with physical exam (91%) and to detect the presence of synovial cysts (68%), whereas tumor/infection (93%) was most likely to cause a change in their approach. In the retrospective review, the physician's planned approach before viewing the MRI was concordant with the actual procedure 49% of the time. A different type of procedure was performed in 15% of planned injections. In such cases, the initial treatment plan was altered (ie, same procedure at a different or additional level or side) in 35% of planned injections. In the prospective data collection, 43% of injections were different from the initial physician decision. The most common reasons for altering the injection was different level affected (36%), facet pathology (22%), and different nerve root affected (16%). CONCLUSIONS: In clinical practice, MRI before injection frequently changes management decisions in the planning and delivery of lumbar spine injections.

Clinical Course of Motor Deficits from Lumbosacral Radiculopathy Due to Disk Herniation.

BACKGROUND: The clinical course of motor deficits from lumbosacral radiculopathy appears to improve with or without surgery. Strength measurements have been confined to manual muscle testing (MMT) and have not been extensively followed and quantified in prior studies. OBJECTIVE: To determine if motor weakness and patient-reported outcomes related to lumbosacral radiculopathy improve without surgical intervention over the course of 12 months. DESIGN: Prospective observational cohort. SETTING: Outpatient academic spine practice. PARTICIPANTS: Adults with acute radicular weakness due to disk herniation. METHODS: Forty patients with radiculopathy and strength deficit were followed over a 12-month period. Objective strength and performance tests as well as survey-based measurements were collected at baseline and then every 3 months. Patients underwent comprehensive pain management and rehabilitation and/or surgical approaches as determined in coordination with the treating specialist. This study was approved by the institutional review board of Colorado. MAIN OUTCOME MEASUREMENTS: Testing of strength was through MMT, handheld dynamometer, and performance-based testing. Furthermore, visual analog scale, modified Oswestry Disability Index, and 36-Item Short Form Health Survey (SF-36) were used to measure pain and disability outcomes. RESULTS: Of the 40 patients, 33 (82.5%) did not have surgery; 7 (17.5%) had surgery. Twenty-four of the 33 patients (60%) did not undergo surgery and were followed for 12 months (Comprehensive Pain Management and Rehabilitation, Complete [CPM&R-C]), and 9 (22%) did not have surgery and lacked at least one follow-up evaluation (Comprehensive Pain Management and Rehabilitation, Incomplete [CPM&R-I]). No statistically significant differences were found on baseline measures of strength deficits and SF-36 domains between the CPM&R-C, Surgery, and CPM&R-I groups. Pain and disability scores in the Surgery group were significantly higher than in the CPM&R-C at baseline. There were statistically significant improvements in all areas of strength, pain, and function when comparing measurements at the 12-month follow-up to baseline in the CPM&R-C group. CONCLUSIONS: Individuals with motor deficits due to lumbosacral radiculopathy improve over time regardless of treatment choice. Most did not choose surgery, and almost all of these patients regained full strength at 1 year. Strength recovery typically occurred in the first 3 months, but there was ongoing recovery over the course of a year. LEVEL OF EVIDENCE: II.

Thermal Stabilization of Biologics with Photoresponsive Hydrogels.

Modern medicine, biological research, and clinical diagnostics depend on the reliable supply and storage of complex biomolecules. However, biomolecules are inherently susceptible to thermal stress and the global distribution of value-added biologics, including vaccines, biotherapeutics, and Research Use Only (RUO) proteins, requires an integrated cold chain from point of manufacture to point of use. To mitigate reliance on the cold chain, formulations have been engineered to protect biologics from thermal stress, including materials-based strategies that impart thermal stability via direct encapsulation of the molecule. While direct encapsulation has demonstrated pronounced stabilization of proteins and complex biological fluids, no solution offers thermal stability while enabling facile and on-demand release from the encapsulating material, a critical feature for broad use. Here we show that direct encapsulation within synthetic, photoresponsive hydrogels protected biologics from thermal stress and afforded user-defined release at the point of use. The poly(ethylene glycol) (PEG)-based hydrogel was formed via a bioorthogonal, click reaction in the presence of biologics without impact on biologic activity. Cleavage of the installed photolabile moiety enabled subsequent dissolution of the network with light and release of the encapsulated biologic. Hydrogel encapsulation improved stability for encapsulated enzymes commonly used in molecular biology (ß-galactosidase, alkaline phosphatase, and T4 DNA ligase) following thermal stress. ß-galactosidase and alkaline phosphatase were stabilized for 4 weeks at temperatures up to 60 °C, and for 60 min at 85 °C for alkaline phosphatase. T4 DNA ligase, which loses activity rapidly at moderately elevated temperatures, was protected during thermal stress of 40 °C for 24 h and 60 °C for 30 min. These data demonstrate a general method to employ reversible polymer networks as robust excipients for thermal stability of complex biologics during storage and shipment that additionally enable on-demand release of active molecules at the point of use.

Development of a cellularly degradable PEG hydrogel to promote articular cartilage extracellular matrix deposition.

Healing articular cartilage remains a significant clinical challenge because of its limited self-healing capacity. While delivery of autologous chondrocytes to cartilage defects has received growing interest, combining cell-based therapies with scaffolds that capture aspects of native tissue and promote cell-mediated remodeling could improve outcomes. Currently, scaffold-based therapies with encapsulated chondrocytes permit matrix production; however, resorption of the scaffold does not match the rate of production by cells leading to generally low extracellular matrix outputs. Here, a poly (ethylene glycol) (PEG) norbornene hydrogel is functionalized with thiolated transforming growth factor (TGF-ß1) and cross-linked by an MMP-degradable peptide. Chondrocytes are co-encapsulated with a smaller population of mesenchymal stem cells, with the goal of stimulating matrix production and increasing bulk mechanical properties of the scaffold. The co-encapsulated cells cleave the MMP-degradable target sequence more readily than either cell population alone. Relative to non-degradable gels, cellularly degraded materials show significantly increased glycosaminoglycan and collagen deposition over just 14 d of culture, while maintaining high levels of viability and producing a more widely-distributed matrix. These results indicate the potential of an enzymatically degradable, peptide-functionalized PEG hydrogel to locally influence and promote cartilage matrix production over a short period. Scaffolds that permit cell-mediated remodeling may be useful in designing treatment options for cartilage tissue engineering applications.

A Biosynthetic Scaffold that Facilitates Chondrocyte-Mediated Degradation and Promotes Articular Cartilage Extracellular Matrix Deposition.

Articular cartilage remains a significant clinical challenge to repair because of its limited self-healing capacity. Interest has grown in the delivery of autologous chondrocytes to cartilage defects, and combining cell-based therapies with scaffolds that capture aspects of native tissue and allow cell-mediated remodeling could improve outcomes. Currently, scaffold-based therapies with encapsulated chondrocytes permit matrix production; however, resorption of the scaffold often does not match the rate of matrix production by chondrocytes, which can limit functional tissue regeneration. Here, we designed a hybrid biosynthetic system consisting of poly (ethylene glycol) (PEG) endcapped with thiols and crosslinked by norbornene-functionalized gelatin via a thiol-ene photopolymerization. The protein crosslinker was selected to facilitate chondrocyte-mediated scaffold remodeling and matrix deposition. Gelatin was functionalized with norbornene to varying degrees (~4-17 norbornenes/gelatin), and the shear modulus of the resulting hydrogels was characterized (<0.1-0.5 kPa). Degradation of the crosslinked PEG-gelatin hydrogels by chondrocyte-secreted enzymes was confirmed by gel permeation chromatography. Finally, chondrocytes encapsulated in these biosynthetic scaffolds showed significantly increased glycosaminoglycan deposition over just 14 days of culture, while maintaining high levels of viability and producing a distributed matrix. These results indicate the potential of a hybrid PEG-gelatin hydrogel to permit chondrocyte-mediated remodeling and promote articular cartilage matrix production. Tunable scaffolds that can easily permit chondrocyte-mediated remodeling may be useful in designing treatment options for cartilage tissue engineering applications.

Covalently tethered TGF-ß1 with encapsulated chondrocytes in a PEG hydrogel system enhances extracellular matrix production.

Healing articular cartilage defects remains a significant clinical challenge because of its limited capacity for self-repair. While delivery of autologous chondrocytes to cartilage defects has received growing interest, combining cell-based therapies with growth factor delivery that can locally signal cells and promote their function is often advantageous. We have previously shown that PEG thiol-ene hydrogels permit covalent attachment of growth factors. However, it is not well known if embedded chondrocytes respond to tethered signals over a long period. Here, chondrocytes were encapsulated in PEG hydrogels functionalized with transforming growth factor-beta 1 (TGF-ß1) with the goal of increasing proliferation and matrix production. Tethered TGF-ß1 was found to be distributed homogenously throughout the gel, and its bioactivity was confirmed with a TGF-ß1 responsive reporter cell line. Relative to solubly delivered TGF-ß1, chondrocytes presented with immobilized TGF-ß1 showed significantly increased DNA content, and GAG and collagen production over 28 days, while maintaining markers of articular cartilage. These results indicate the potential of thiol-ene chemistry to covalently conjugate TGF-ß1 to PEG to locally influence chondrocyte function over 4 weeks. Scaffolds with other or multiple tethered growth factors may prove broadly useful in the design of chondrocyte delivery vehicles for cartilage tissue engineering applications.

Alignment of multi-layered muscle cells within three-dimensional hydrogel macrochannels.

This work describes the development and testing of poly(ethylene glycol) (PEG) hydrogels with independently controlled dimensions of wide and deep macrochannels for their ability to promote alignment of skeletal myoblasts and myoblast differentiation. A UV-photopatterned thiol-ene mold was employed to produce long channels, which ranged from ∼40 to 200 µm in width and from ∼100 to 200 µm in depth, within a PEG-RGD hydrogel. Skeletal myoblasts (C2C12) were successfully cultured multiple cell layers deep within the channels. Decreasing channel width, increasing channel depth and, interestingly, increasing cell layer away from the channel base all contributed to a decreased interquartile range of cell angle relative to the long axis of the channel wall, indicating improved cell alignment. Differentiation of skeletal myoblasts into myotubes was confirmed by gene expression for myoD, myogenin and MCH IIb, and myotube formation for all channel geometries, but was not dependent on channel size. Qualitatively, myotubes were characteristically different, as myotubes were larger and had more nuclei in larger channels. Overall, our findings demonstrate that relatively large features, which do not readily facilitate cell alignment in two dimensions, promote cell alignment when presented in three dimensions, suggesting an important role for three-dimensional spatial cues.