Bone Marrow Mobilization and Local Stromal Cell-Derived Factor-1α Delivery Enhances Nascent Supraspinatus Muscle Fiber Growth.

Rotator cuff tear is a significant problem that leads to poor clinical outcomes due to muscle degeneration after injury. The objective of this study was to synergistically increase the number of proregenerative cells recruited to injure rotator cuff muscle through a novel dual treatment system, consisting of a bone marrow mobilizing agent (VPC01091), hypothesized to "push" prohealing cells into the blood, and localized delivery of stromal cell-derived factor-1α (SDF-1α), to "pull" the cells to the injury site. Immediately after rotator cuff tendon injury in rat, the mobilizing agent was delivered systemically, and SDF-1α-loaded heparin-based microparticles were injected into the supraspinatus muscle. Regenerative and degenerative changes to supraspinatus muscle and the presence of inflammatory/immune cells, mesenchymal stem cells (MSCs), and satellite cells were assessed via flow cytometry and histology for up to 21 days. After dual treatment, significantly more MSCs (31.9 ± 8.0% single cells) and T lymphocytes (6.7 ± 4.3 per 20 × field of view) were observed in supraspinatus muscle 7 days after injury and treatment compared to injury alone (14.4 ± 6.5% single cells, 1.2 ± 0.7 per 20 × field of view), in addition to an elevated M2:M1 macrophage ratio (3.0 ± 0.5), an indicator of a proregenerative environment. These proregenerative cellular changes were accompanied by increased nascent fiber formation (indicated by embryonic myosin heavy chain staining) at day 7 compared to SDF-1α treatment alone, suggesting that this method may be a promising strategy to influence the early cellular response in muscle and promote a proregenerative microenvironment to increase muscle healing after severe rotator cuff tear.

Microfluidic Platform for Microparticle Fabrication and Release of a Cathepsin Inhibitor.

Cathepsins are a family of cysteine proteases responsible for a variety of homeostatic functions throughout the body, including extracellular matrix remodeling, and have been implicated in a variety of degenerative diseases. However, clinical trials using systemic administration of cathepsin inhibitors have been abandoned due to side effects, so local delivery of cathepsin inhibitors may be advantageous. In these experiments, a novel microfluidic device platform was developed that can synthesize uniform, hydrolytically degradable microparticles from a combination of poly(ethylene glycol) diacrylate (PEGDA) and dithiothreitol (DTT). Of the formulations examined, the 10-polymer weight percentage 10 mM DTT formulation degraded after 77 days in vitro. A modified assay using the DQ Gelatin Fluorogenic Substrate was used to demonstrate sustained release and bioactivity of a cathepsin inhibitor (E-64) released from hydrogel microparticles over 2 weeks in vitro (up to ∼13 µg/mL released with up to ∼40% original level of inhibition remaining at day 14). Altogether, the technologies developed in this study will allow a small-molecule, broad cathepsin inhibitor E-64 to be released in a sustained manner for localized inhibition of cathepsins for a wide variety of diseases.

Development and characterization of Factor Xa-responsive materials for applications in cell culture and biologics delivery.

Stimuli-responsive biomaterials may be used to better control the release of bioactive molecules or cells for applications involving drug delivery and controlled cell release. In this study, we developed a Factor Xa (FXa)-responsive biomaterial capable of controlled release of pharmaceutical agents and cells from in vitro culture. FXa-cleavable substrates were formed as hydrogels that degraded in response to FXa enzyme over several hours. Hydrogels were shown to release both heparin and a model protein in response to FXa. Additionally, RGD-functionalized FXa-degradable hydrogels were used to culture mesenchymal stromal cells (MSCs), enabling FXa-mediated cell dissociation from hydrogels in a manner that preserved multicellular structures. Harvesting MSCs using FXa-mediated dissociation did not influence their differentiation capacity or indoleamine 2,3-dioxygenase (IDO) activity (a measure of immunomodulatory capacity). In all, this FXa-degradable hydrogel is a novel responsive biomaterial system that may be used for on-demand drug delivery, as well as for improving processes for in vitro culture of therapeutic cells.

Growth Factor Immobilization Strategies for Musculoskeletal Disorders.

PURPOSE OF REVIEW: Tissue regenerative solutions for musculoskeletal disorders have become increasingly important with a growing aged population. Current growth factor treatments often require high dosages with the potential for off-target effects. Growth factor immobilization strategies offer approaches towards alleviating these concerns. This review summarizes current growth factor immobilization techniques (encapsulation, affinity interactions, and covalent binding) and the effects of immobilization on growth factor loading, release, and bioactivity. RECENT FINDINGS: The breadth of immobilization techniques based on encapsulation, affinity, and covalent binding offer multiple methods to improve the therapeutic efficacy of growth factors by controlling bioactivity and release. Growth factor immobilization strategies have evolved to more complex systems with the capacity to load and release multiple growth factors with spatiotemporal control. The advancements in immobilization strategies allow for development of new, complex musculoskeletal tissue treatment strategies with improved spatiotemporal control of loading, release, and bioactivity.

Injection of Micronized Human Amnion/Chorion Membrane Results in Increased Early Supraspinatus Muscle Regeneration in a Chronic Model of Rotator Cuff Tear.

Surgical repair of severe rotator cuff tear often results in retear due to unaddressed muscle degeneration. The objective of this study was to test the regenerative potential of micronized dehydrated Human Amnion/Chorion Membrane (dHACM), in a clinically relevant delayed reattachment model of rotator cuff repair. Micronized dHACM was injected into rat supraspinatus muscle during tendon re-attachment surgery, three weeks after original tendon injury. One week after material injection, inflammatory and mesenchymal stem cell infiltration into supraspinatus muscles was assessed via flow cytometry. Histological methods were utilized to assess structural and regenerative changes in muscle one and three weeks after material injection. Micronized dHACM injection resulted in increased M1-like macrophages (17.1 [Formula: see text] fold change over contralateral controls) and regenerating muscle fibers (4.3% vs 1.7% in saline treated muscles) one week after injection compared to saline treated muscles. Tendon reattachment itself exhibited intrinsic healing in this model, demonstrated by a general return of muscle weight and reduced fibrosis. Our results indicate that injection of micronized dHACM may initiate an inflammatory response in degenerated muscle that promotes early muscle regeneration, and that our animal model may be a suitable platform for studying treatments in muscle at early timepoints, before intrinsic healing occurs.

In vivo hydroxyapatite scaffold performance in infected bone defects.

Critically sized bone defects are often compounded by infectious complications. The standard of care consists of bone autografts with systemic antibiotics. These injuries and treatments lead to donor site morbidity, antibiotic resistant strains of bacteria, and often end stage amputation. This study proposes an alternative to the autograft using a porous, hydroxyapatite (HA) scaffold evaluated with and without infection and antibiotics. Twenty-four New Zealand white rabbits received either our HA scaffold or a pulverized autograft (PBA) within a surgically created critical-sized defect in the femur. The two grafts were evaluated in either septic or aseptic defects and with or without antibiotic treatment. The HA scaffolds were characterized with micro computed tomography. Post-euthanasia, micro computed tomography, histology, and white blood cells component analysis were completed. The HA had significantly greater (p < .001) mineralization to total volume than the PBA groups with 27.56% and 14.88%, respectively, and the septic HA groups were significantly greater than the aseptic groups both with and without antibiotics (p = .016). The bone quality denoted by bone mineral density was also significantly greater (p < .001) in the HA groups (67.01 ± 0.38 mgHA/cm3 ) than the PBA groups (64.66 ± 0.85 mgHA/cm3 ). The HA scaffold is a viable alternative to the bone autograft in defects with and without infection as shown by the quality and quantity of bone.

Connexin 43 hemichannels protect bone loss during estrogen deficiency.

Estrogen deficiency in postmenopausal women is a major cause of bone loss, resulting in osteopenia, osteoporosis, and a high risk for bone fracture. Connexin 43 (Cx43) hemichannels (HCs) in osteocytes play an important role in osteocyte viability, bone formation, and remodeling. We showed here that estrogen deficiency reduced Cx43 expression and HC function. To determine if functional HCs protect osteocytes and bone loss during estrogen deficiency, we adopted an ovariectomy model in wild-type (WT) and two transgenic Cx43 mice: R76W (dominant-negative mutant inhibiting only gap junction channels) and Cx43 Δ130-136 (dominant-negative mutant compromising both gap junction channels and HCs). The bone mineral density (BMD), bone structure, and histomorphometric changes of cortical and trabecular bones after ovariectomy were investigated. Our results showed that the Δ130-136 transgenic cohort had greatly decreased vertebral trabecular bone mass compared to WT and R76W mice, associated with a significant increase in the number of apoptotic osteocyte and empty lacunae. Moreover, osteoclast surfaces in trabecular and cortical bones were increased after ovariectomy in the R76W and WT mice, respectively, but not in ∆130-136 mice. These data demonstrate that impairment of Cx43 HCs in osteocytes accelerates vertebral trabecular bone loss and increase in osteocyte apoptosis, and further suggest that Cx43 HCs in osteocytes protect trabecular bone against catabolic effects due to estrogen deficiency.

Quantification of injection force mechanics during injection laryngoplasty.

OBJECTIVES: In-office or operative injection laryngoplasty requires needle stability for accurate material placement. To date, no reports compare injection forces based on needle gauge, bends, length, or material type or temperature. We hypothesize these factors alter injection forces and could impact clinical use. METHODS: Swine larynges were placed in a compression testing machine. Syringes were affixed to a stabilizing crossbeam. Straight needles (25G 1.5-inch; 27G 1.25-inch; or 9.8-inch malleable shaft 16G per oral with 24G tapered needle tip) were inserted into the swine vocal folds to simulate realistic tissue resistance pressure. Compressive loading was conducted at 40 mm/minute until steady-state force was achieved. Tests were completed with calcium hydroxylapatite (CaHa), carboxymethylcellulose, and hyaluronic acid at various temperatures and CaHa with various bends in the needles (n = 3 per group, comparisons performed by two-way analysis of variance (ANOVA), Tukey's post-hoc). RESULTS: Needle size, shape, and temperature altered injection force. Steady-state force was highest with the per-oral needle at a mean of 44.55N compared to 26.44N and 29.77N in the 25G and 27G percutaneous needles, respectively (P < 0.001). Stiffness rate (initial increasing force vs. distance to initiate injection) ranged from 19.75N/mm (per oral) to 22.06N/mm (25G) to 24.56N/mm (27G), (P = 0.875). Adding multiple bends to the per-oral needle increased stiffness rate to 24.99N/mm (P = 0.035), whereas the 25G needle stiffness rate remained unchanged (P = 0.941), with the stiffness rate decreasing in the 27G needle with increasing bends (P = 0.033). Increased temperature decreased injection forces across all materials. CONCLUSION: Needle caliber, length, and bends impact steady-state forces and stiffness rates during vocal fold injection. LEVEL OF EVIDENCE: NA Laryngoscope, 129:1060-1066, 2019.

Three-dimensional printing for craniomaxillofacial regeneration.

Craniomaxillofacial injuries produce complex wound environments involving various tissue types and treatment strategies. In a clinical setting, care is taken to properly irrigate and stabilize the injury, while grafts are molded in an attempt to maintain physiological functionality and cosmesis. This often requires multiple surgeries and grafts leading to added discomfort, pain and financial burden. Many of these injuries can lead to disfigurement and resultant loss of system function including mastication, respiration, and articulation, and these can lead to acute and long-term psychological impact on the patient. A main causality of these issues is the lack of an ability to spatially control pre-injury morphology while maintaining shape and function. With the advent of additive manufacturing (three-dimensional printing) and its use in conjunction with biomaterial regenerative strategies and stem cell research, there is an increased potential capacity to alleviate such limitations. This review focuses on the current capabilities of additive manufacturing platforms, completed research and potential for future uses in the treatment of craniomaxillofacial injuries, with an in-depth discussion of regeneration of the periodontal complex and teeth.

An in vitro fluid dynamic study of pediatric cannulae: the value of animal studies to predict human flow.

A challenge to the development of pediatric ventricular assist devices (PVADs) is the use of the aortic cannulae attached to the devices. Cannulae used for pediatric application have small diameters and large pressure drops. Furthermore, during the development of the 12cc Penn State pediatric PVAD, particle image velocimetry (PIV) illustrated that hematocrit levels, through changes in blood viscoelasticity, affected the fluid dynamics. The objective of this study is to compare the fluid dynamics of a pediatric viscoelastic blood analog and a goat viscoelastic blood analog within the PVAD aortic cannula. Two acrylic models were manufactured to model the aortic cannula (6 mm and 8 mm diameters). PIV data was collected to examine the flow at the outlet of the VAD and in the aortic cannula at heart rates of 50 and 75 beats per minute (bpm). Three planes of data were taken, one at the centerline and two 1.5 mm above and below the centerline. Three more planes of data were taken orthogonal to the original planes. While a 75 bpm heart rate was used to represent normal operating conditions, a 50 bpm heart rate represented use of the PVAD during weaning. At 75 bpm, differences were evident between the two different fluids and the two models. Separation zones developed in the plane below the centerline for the higher hematocrit pediatric blood analog. This study raises question to the usefulness of animal testing results in regard to how well they predict the outcome of pediatric patients.