The influence of radiation-induced collagen chain fragmentation, crosslinking, and sequential irradiation on the high-cycle fatigue life of human cortical bone.

Both high-cycle fatigue life and fatigue crack propagation resistance of human cortical bone allograft are radiation dose-dependent between 0 and 25 kGy such that higher doses exhibit progressively shorter lifetimes. Recently, we have shown that collagen chain fragmentation and stable crosslink accumulation may contribute to the radiation dose-dependent loss in fatigue crack propagation resistance of human cortical bone. To our knowledge, the influence of these mechanisms on high-cycle fatigue life of cortical bone have not been established. Sequential irradiation has also been shown to mitigate the loss of fatigue life of tendons, however, whether this mitigates losses in fatigue life of cortical bone has not been explored. Our objectives were to evaluate the influence of radiation-induced collagen chain fragmentation and crosslinking on the high-cycle fatigue life of cortical bone in the dose range of 0-15 kGy, and to evaluate the capability of sequential irradiation at 15 kGy to mitigate the loss of high-cycle fatigue life and radiation-induced collagen damage. High-cycle fatigue life specimens from four male donor femoral pairs were divided into 5 treatment groups (0 kGy, 5 kGy, 10 kGy, 15 kGy, and 15 kGy sequentially irradiated) and subjected to high-cycle fatigue life testing with a custom rotating-bending apparatus at a cyclic stress of 35 MPa. Following fatigue testing, collagen was isolated from fatigue specimens, and collagen chain fragmentation and crosslink accumulation were quantified using SDS-PAGE and a fluorometric assay, respectively. Both collagen chain fragmentation (p = 0.006) and non-enzymatic crosslinking (p < 0.001) influenced high-cycle fatigue life, which decreased with increasing radiation dose from 0 to 15 kGy (p = 0.016). Sequential irradiation at 15 kGy did not offer any mitigation in high-cycle fatigue life (p = 0.93), collagen chain fragmentation (p = 0.99), or non-enzymatic crosslinking (p ≥ 0.10) compared to a single radiation dose of 15 kGy. Taken together with our previous findings on the influence of collagen damage on fatigue crack propagation resistance, collagen chain fragmentation and crosslink accumulation both contribute to radiation-induced losses in notched and unnotched fatigue life of cortical bone. To maximize the functional lifetime of radiation sterilized structural cortical bone allografts, pathways other than sequential radiation should be explored to mitigate collagen matrix damage.

Differential effects of macrophage subtype-specific cytokines on fibroblast proliferation and endothelial cell function in co-culture system.

Macrophages are involved in several critical activities associated with tissue repair and regeneration. Current approaches in regenerative medicine are focusing on leveraging the innate immune response to accelerate tissue regeneration and improve long-term healing outcomes. Of particular interest in this regard are the currently known, four main M2 macrophage subtypes: M2interleukin (IL)-4,IL-13, M2IC, M2IL-10, M2non-selective adenosine receptor agonists (NECA) (M2IL-4,IL-13 → M2NECA). In this study, rat bone marrow-derived macrophages (M0) were polarized to each of the four subtypes M2IL-4,IL-13 → M2NECA and cultured for 72 h in vitro. Luminex assay results highlighted increased production of tissue inhibitor of metalloproteinases-1 (TIMP-1) for M2IL-4,IL-13, higher amounts of transforming growth factor-beta 1 (TGF-ß1) for M2IL-10, and elevated vascular endothelial growth factor A (VEGF-A) from M2NECA. Co-culture experiments performed with M2IL-10 macrophages and L929 fibroblasts highlighted the increased production of soluble collagen within the media as well as higher amounts of collagen in the extracellular matrix. Human umbilical vein endothelial cells (HUVECs) were co-cultured with M2NECA macrophages, which demonstrated an increase in intercellular adhesion molecule (ICAM) and platelet endothelial cell adhesion molecule (PECAM), as well as increased formation of endothelial tubes. The findings of this study emphasize a critical demand for further characterization and analyses of distinct M2 subtypes and careful selection of specific macrophage populations for regeneration of specific tissue types. The current, broad classification of "M2" may be sufficient in many general tissue engineering applications, but, as conditions are constantly in flux within the microenvironment in vivo, a higher degree of specificity and control over the initial M2 subtype could result in more consistent long-term outcomes where macrophages are utilized as part of an overall regenerative strategy.

Dose-dependent effects of gamma radiation sterilization on the collagen matrix of human cortical bone allograft and its influence on fatigue crack propagation resistance.

Fatigue crack propagation resistance and high-cycle S-N fatigue life of cortical bone allograft tissue are both negatively impacted in a radiation dose-dependent manner from 0 to 25 kGy. The standard radiation sterilization dose of 25-35 kGy has been shown to induce cleavage of collagen molecules into smaller peptides and accumulation of stable crosslinks within the collagen matrix, suggesting that these mechanisms may influence radiation-induced losses in cyclic fracture resistance. The objective of this study was to determine the radiation dose-dependency of collagen chain fragmentation and crosslink accumulation within the dose range of 0-25 kGy. Previously, cortical bone compact tension specimens from two donor femoral pairs were divided into four treatment groups (0 kGy, 10 kGy, 17.5 kGy, and 25 kGy) and underwent cyclic loading fatigue crack propagation testing. Following fatigue testing, collagen was isolated from one compact tension specimen in each treatment group from both donors. Radiation-induced collagen chain fragmentation was assessed using SDS-PAGE (n = 5), and accumulation of pentosidine, pyridinoline, and non-specific advanced glycation end products were assessed using a fluorometric assay (n = 4). Collagen chain fragmentation increased progressively in a dose-dependent manner (p < 0.001). Crosslink accumulation at all radiation dose levels increased relative to the 0 kGy control but did not demonstrate dose-dependency (p < 0.001). Taken together with our previous findings on fatigue crack propagation behavior, these data suggest that while collagen crosslink accumulation may contribute to reduced notched fatigue behavior with irradiation, dose-dependent losses in fatigue crack propagation resistance are mainly influenced by radiation-induced chain fragmentation.

Nodes for modes: nodal honeycomb metamaterial enables a soft robot with multimodal locomotion.

Soft-bodied animals, such as worms and snakes, use many muscles in different ways to traverse unstructured environments and inspire tools for accessing confined spaces. They demonstrate versatility of locomotion which is essential for adaptation to changing terrain conditions. However, replicating such versatility in untethered soft-bodied robots with multimodal locomotion capabilities have been challenging due to complex fabrication processes and limitations of soft body structures to accommodate hardware such as actuators, batteries and circuit boards. Here, we present MetaCrawler, a 3D printed metamaterial soft robot designed for multimodal and omnidirectional locomotion. Our design approach facilitated an easy fabrication process through a discrete assembly of a modular nodal honeycomb lattice with soft and hard components. A crucial benefit of the nodal honeycomb architecture is the ability of its hard components, nodes, to accommodate a distributed actuation system, comprising servomotors, control circuits, and batteries. Enabled by this distributed actuation, MetaCrawler achieves five locomotion modes: peristalsis, sidewinding, sideways translation, turn-in-place, and anguilliform. Demonstrations showcase MetaCrawler's adaptability in confined channel navigation, vertical traversing, and maze exploration. This soft robotic system holds the potential to offer easy-to-fabricate and accessible solutions for multimodal locomotion in applications such as search and rescue, pipeline inspection, and space missions.

Rapid measurement of hemoglobin-oxygen dissociation by leveraging Bohr effect and Soret band bathochromic shift.

Oxygen (O2) binds to hemoglobin (Hb) in the lungs and is then released (dissociated) in the tissues. The Bohr effect is a physiological mechanism that governs the affinity of Hb for O2 based on pH, where a lower pH results in a lower Hb-O2 affinity and higher Hb-O2 dissociation. Hb-O2 affinity and dissociation are crucial for maintaining aerobic metabolism in cells and tissues. Despite its vital role in human physiology, Hb-O2 dissociation measurement is underutilized in basic research and in clinical laboratories, primarily due to the technical complexity and limited throughput of existing methods. We present a rapid Hb-O2 dissociation measurement approach by leveraging the Bohr effect and detecting the optical shift in the Soret band that corresponds to the light absorption by the heme group in Hb. This new method reduces Hb-O2 dissociation measurement time from hours to minutes. We show that Hb deoxygenation can be accelerated chemically at the optimal pH of 6.9. We show that time and pH-controlled deoxygenation of Hb results in rapid and distinct conformational changes in its tertiary structure. These molecular conformational changes are manifested as significant, detectable shifts in Hb's optical absorption spectrum, particularly in the characteristic Soret band (414 nm). We extensively validated the method by testing human blood samples containing normal Hb and Hb variants. We show that rapid Hb-O2 dissociation can be used to screen for and detect Hb-O2 affinity disorders and to evaluate the function and efficacy of Hb-modifying therapies. The ubiquity of optical absorption spectrophotometers positions this approach as an accessible, rapid, and accurate Hb-O2 dissociation measurement method for basic research and clinical use. We anticipate this method's broad adoption will democratize the diagnosis and prognosis of Hb disorders, such as sickle cell disease. Further, this method has the potential to transform the research and development of new targeted and genome-editing-based therapies that aim to modify or improve Hb-O2 affinity.

Immature porcine cortical bone mechanical properties and composition change with maturation and displacement rate.

Computational models of mature bone have been used to predict fracture; however, analogous study of immature diaphyseal fracture has not been conducted due to sparse experimental mechanical data. A model of immature bone fracture may be used to aid in the differentiation of accidental and non-accidental trauma fractures in young, newly ambulatory children (0-3 years). The objective of this study was to characterize the evolution of tissue-level mechanical behavior, composition, and microstructure of maturing cortical porcine bone with uniaxial tension, Raman spectroscopy, and light microscopy as a function of maturation. We asked: 1) How do the monotonic uniaxial tensile properties change with maturation and displacement rate; 2) How does the composition and microstructure change with maturation; and 3) Is there a correlation between composition and tensile properties with maturation? Elastic modulus (p < 0.001), fracture stress (p < 0.001), and energy absorption (p < 0.014) increased as a function of maturation at the quasistatic rate by 110%, 86%, and 96%, respectively. Fracture stress also increased by 90% with maturation at the faster rate (p = 0.001). Fracture stress increased as a function of increasing displacement rate by 28% (newborn p = 0.048; 1-month p = 0.004; 3-month p= < 0.001), and fracture strain decreased by 68% with increasing displacement rate (newborn p = 0.002; 1-month p = 0.036; 3-month p < 0.001). Carbonate-to-phosphate ratio was positively linearly related to elastic modulus, and fracture stress was positively related to carbonate-to-phosphate ratio and matrix maturation ratio. The results of this study support that immature bone is strain-rate dependent and becomes more brittle at faster rates, contributing to the foundation upon which a computational model can be built to evaluate immature bone fracture.

Dexamethasone eluting polydopaminated polycaprolactone-poly (lactic-co-glycolic) acid for treatment of tracheal stenosis.

Tracheal stenosis is commonly caused by injury, resulting in inflammation and fibrosis. Inhibiting inflammation and promoting epithelization can reduce recurrence after initial successful treatment of tracheal stenosis. Steroids play an important role in tracheal stenosis management. This study in vitro evaluated effectiveness of a polydopaminated polycaprolactone stent coated with dexamethasone-eluting poly(lactic-co-glycolic) acid microparticles (µPLGA) for tracheal stenosis management. Polydopamination was characterized by Raman spectroscopy and promoted epithelialization while dexamethasone delivery reduced macrophage activity, assessed by individual cell area measurements and immunofluorescent staining for inducible nitric oxide synthase (iNOS). Dexamethasone release was quantified by high-performance liquid chromatography over 30 days. Activation-related increase in cell area and iNOS production by RAW 264.7 were both reduced significantly (p < .05) through dexamethasone release. Epithelial cell spreading was higher on polydopaminated polycaprolactone (PCL) than PCL-alone (p < .05). Force required for stent migration was measured by pullout tests of PCL-µPLGA stents from cadaveric rabbit and porcine tracheas (0.425 ± 0.068 N and 1.082 ± 0.064 N, respectively) were above forces estimated to occur during forced respiration. Biomechanical support provided by stents to prevent airway collapse was assessed by comparing compressive circumferential stiffness, and stiffness of the stent was about 1/10th of the rabbit trachea (0.156 ± 0.023 N/mm vs. 1.420 ± 0.194 N/mm, respectively). A dexamethasone-loaded PCL-µPLGA stent platform can deliver dexamethasone and exhibits sufficient mechanical properties to anchor within the trachea and polydopamination of PCL is conducive to epithelial layer formation. Therefore, a polydopaminated PCL-µPLGA stent is a promising candidate for in vivo evaluation for treatment of tracheal restenosis.

Fatigue crack propagation and fracture toughness of cortical bone are radiation dose-dependent.

Cortical bone allograft sterilized with a standard γ-radiation dose of 25-35kGy has demonstrated reduced static and cyclic fracture resistance compared with unirradiated bone. To mitigate radiation damage, we recently observed a dose-dependent response of high-cycle fatigue behavior of human cortical bone from 0 to 25 kGy, with lower doses exhibiting logarithmically longer fatigue lives. The objectives of this study were as follows: (1) to determine whether fracture toughness, work-to-fracture, and fatigue crack propagation resistance of human cortical bone are also radiation dose-dependent, and (2) to determine the associations of radiation dose and a Raman biomarker for collagen disorder with fracture properties. Compact tension specimens were machined from two donor femoral pairs and allocated to four treatment groups: 0 (unirradiated control), 10, 17.5, and 25 kGy. Fracture toughness specimens were monotonically loaded to failure and the critical stress intensity factor (KC ) was determined. Work-to-fracture was calculated from the load versus displacement integral up to fracture. Fatigue crack propagation specimens were cyclically loaded under constant room-temperature irrigation and fatigue crack growth rate (da/dN) and cyclic stress intensity (∆K) were calculated. Fracture toughness, work-to-fracture, and fatigue crack propagation resistance decreased 18%, 33%, and 15-fold from 0 to 25 kGy, respectively (p < 0.05). Radiation dose was more predictive of fracture properties than collagen disorder. These findings support that quasi-static and fatigue fracture properties of cortical bone are radiation dose-dependent within this dose range. The structural alterations arising from irradiation that cause these losses in fracture resistance remain to be elucidated.

Comparison of Morphological and Histological Characteristics of Human and Sheep: Sheep as a Potential Model for Testing Midurethral Slings in vivo.

INTRODUCTION: The sheep was evaluated as a potential model for preclinical evaluation of urethral slings in vivo based on: (1) anatomical measurements of the sheep vagina and (2) histological tissue integration and host response to polypropylene (PP) slings. METHODS: Eight female, multiparous sheep were utilized. Three of 8 animals underwent surgery mimicking human tension-free vaginal tape protocols for midurethral slings and were euthanized at 6 months. The following measurements were obtained: vaginal length, maximum vaginal width with retraction, symphysis pubis length, and distance from the pubic bone to incision. Explanted sling samples from sheep and human were stained with hematoxylin and eosin for host reaction assessment. RESULTS: Geometric measurements were similar between humans and sheep. Sheep vaginal anatomy allowed sling placement similar to procedures in human surgeries, and all sheep recovered without problems. Comparative histology between the sheep and human indicated similar host reaction and collagen deposition around implants, confirming suitability of the sheep model for biomaterial response assessment. CONCLUSION: Sheep vaginal length is comparable to humans. Tissue integration and host response to PP slings showed chronic inflammation with rich collagen deposition around the material in both sheep and human specimens, highlighting the sheep as a potential animal model for preclinical testing of midurethral slings.

Calcium­dependent activation of PHEX, MEPE and DMP1 in osteocytes.

Calcium (Ca2+) signaling is the first messenger signal exhibited by osteocytes. The present study aimed to better understand the link between Ca2+ concentration, and the levels of bone mineralization regulator proteins [phosphate­regulating neutral endopeptidase on chromosome X (PHEX), matrix extracellular phosphoglycoprotein (MEPE) and dentin matrix protein 1 (DMP1)] and the levels of oxidative stress in osteocytes. The viability of MLO­Y4 cells was determined using the live/dead assay following treatment with various Ca2+ concentrations (1.8, 6, 12, 18, 24 and 50 mM) for different durations (15 and 60 min, and 24 h). Superoxide dismutase (SOD), catalase (CAT), glutathione (GSH) and NADPH oxidase (NOX) enzymes were analyzed using a colorimetric method. Apoptosis was detected by caspase­3 analysis. Furthermore, the protein expression levels of PHEX, MEPE and DMP1 were analyzed using immunoblotting, and oxidative stress was examined using the total antioxidant and total oxidant status (TOS) assay. Notably, after 15 min, there were more live cells than dead cells; however, after 60 min, the number of dead cells was increased following treatment with 24 and 50 mM Ca2+. After 24 h, there were more dead cells than live cells following treatment with 50 mM Ca2+. After 24 h of Ca2+ treatment, the highest protein expression levels of PHEX, MEPE and DMP1 were measured in cells treated with 24 mM Ca2+. In addition, as Ca2+ concentration increased, the TOS and the oxidative stress index values were also increased. In conclusion, these results suggested that 24 mM Ca2+ may trigger bone mineralization proteins, such as PHEX, MEPE and DMP1, and could be considered an applicable dosage for the treatment of bone damage in the future.

A preliminary evaluation of in vivo response to a filament-wound macroporous collagen midurethral sling in an ovine model.

Stress urinary incontinence (SUI) impacts ~1/3 of women over age 50. Negative publicity around PP meshes used in pelvic prolapse repair drives the need for identifying alternative biomaterials for SUI repair. Our study evaluated in vivo response to collagen sling implanted in an ovine model. Electrocompacted collagen threads were filament wound as slings and crosslinked in genipin. Collagen slings were implanted suburethrally mimicking the transvaginal tape technique. Main study groups were: Collagen sling (n = 3, 6 months) and PP sling (n = 3, 6 months). Collagen sling was also tested at 3-weeks (n = 1) to observe early-stage tissue response and 1-year (n = 2) to assess biomaterial longevity in a preliminary capacity. Collagen slings healed to a fibrous ligament texture at 6 months and maintained such texture to 1 year. Histological scoring indicated biocompatible responses to collagen slings with no adverse events. All study groups exhibited complete tissue ingrowth and interstitial de novo collagen deposition at all time points. Collagen threads induced orderly de novo collagen deposition that was aligned along long axes of threads. Tissue infiltrated collagen slings that were explanted at 6 and 12 months presented similar structural strength with native tissues such as vagina and fascia, and PP (Lynx) slings (p > .05). With the limitation of low number of animals per time point in hindsight, this preliminary study justifies evaluation of collagen slings in a larger sample size of animals, particularly to assess persistence of ligamentous tissue response over longer durations than 1-year.

A collagen/PLA hybrid scaffold supports tendon-derived cell growth for tendon repair and regeneration.

A rotator cuff tendon tear is a common shoulder injury with a relatively high rate of recurrence after surgical repair. In order to reinforce the repair and reduce the risk of clinical complications, a patch scaffold is typically sutured over the tendon tear to provide post-surgical mechanical support. However, despite considerable research effort in this area, a patch scaffold that provides both superior initial mechanical properties and supports cell proliferation at the same time has not yet been achieved. In this study, we engineered a collagen/poly(lactic acid) (COL/PLA) hybrid yarn to leverage mechanical strength of PLA yarn and the bioactivity of collagen. The COL/PLA yarns were used to fabricate a tissue engineering scaffold using textile weaving technology. This hybrid scaffold had a tensile strength of 354.0 ± 36.0 N under dry conditions and 267.2 ± 15.9 N under wet conditions, which was satisfactory to maintain normal tendon function. By introducing COL yarns into the hybrid scaffold, the proliferation of tendon-derived cells was significantly improved on the scaffold. Cell coverage after 28-days of in vitro cell culture was noticeably higher on the COL yarns compared to the PLA yarns as a result of a larger number of cells and more spread cell morphology on collagen. Cells spread in multiple directions on COL yarns, which resembled a more natural cell attachment on extracellular matrix. On the contrary, the cells attached to the PLA filaments presented an elongated morphology along the fiber's axial direction. Combining the mechanical robustness of PLA and the biological activity of collagen, the woven COL/PLA hybrid scaffold has shown its potential to be a promising candidate for tendon repair applications.

Mesenchymal Stem Cell Delivery via Topographically Tenoinductive Collagen Biotextile Enhances Regeneration of Segmental Tendon Defects.

BACKGROUND: Successful management of massive rotator cuff (RC) tendon tears represents a treatment challenge because of the limited intrinsic healing capacity of native tendons and the risk of repair failure. Biologic augmentation of massive RC tears utilizing scaffolds-capable of regenerating bulk tendon tissue to achieve a mechanically functional repair-represents an area of increasing clinical interest. PURPOSE: To investigate the histological and biomechanical outcomes after the use of a novel biologic scaffold fabricated from woven electrochemically aligned collagen (ELAC) threads as a suture-holding, fully load-bearing, defect-bridging scaffold with or without mesenchymal stem cells (MSCs) compared with direct repair in the treatment of critically sized RC defects using a rabbit model. STUDY DESIGN: Controlled laboratory study. METHODS: A total of 34 New Zealand White rabbits underwent iatrogenic creation of a critically sized defect (6 mm) in the infraspinatus tendon of 1 shoulder, with the contralateral shoulder utilized as an intact control. Specimens were divided into 4 groups: (1) gap-negative control without repair; (2) direct repair of the infraspinatus tendon-operative control; (3) tendon repair using ELAC; and (4) tendon repair using ELAC + MSCs. Repair outcomes were assessed at 6 months using micro-computed tomography, biomechanical testing, histology, and immunohistochemistry. RESULTS: Specimens treated with ELAC demonstrated significantly less tendon retraction when compared with the direct repair group specimens (P = .014). ELAC + MSCs possessed comparable biomechanical strength (178 ± 50 N) to intact control shoulders (199 ± 35 N) (P = .554). Histological analyses demonstrated abundant, well-aligned de novo collagen around ELAC threads in both the ELAC and the ELAC + MSC shoulders, with ELAC + MSC specimens demonstrating increased ELAC resorption (7% vs 37%, respectively; P = .002). The presence of extracellular matrix components, collagen type I, and tenomodulin, indicating tendon-like tissue formation, was appreciated in both the ELAC and the ELAC + MSC groups. CONCLUSION: The application of MSCs to ELAC scaffolds improved biomechanical and histological outcomes when compared with direct repair for the treatment of critically sized defects of the RC in a rabbit model. CLINICAL RELEVANCE: This study demonstrates the feasibility of repairing segmental tendon defects with a load-bearing, collagen biotextile in an animal model, showing the potential applicability of RC repair supplementation using allogeneic stem cells.

In Vivo Delivery of M0, M1, and M2 Macrophage Subtypes via Genipin-Cross-Linked Collagen Biotextile.

Developing strategies to regulate the immune response poses significant challenges with respect to the clinical translation of tissue-engineered scaffolds. Prominent advancements have been made relating to macrophage-based therapies and biomaterials. Macrophages exhibit the potential to influence healing trajectory, and predominance of particular subtypes during early onset of healing influences repair outcomes. This study evaluated short- and long-term healing response and postoperative mechanical properties of genipin-cross-linked, electrochemically aligned collagen biotextiles with comparative administration of M0, M1, and M2 subtypes. Irrespective of macrophage subtype seeded, all the groups demonstrated existence of M2 macrophages at both time points as typified by arginase and Ym-1 expressions, and distinct absence of M1 macrophages, as indicated by lack of inducible nitric oxide synthase (iNOS) and interleukin-1ß expression in all the groups for both time points. M2 macrophage-seeded collagen biotextiles revealed promising host tissue responses, such as reduced fibrous capsule thickness and minimal granulation tissue formation. Furthermore, the M2-seeded group displayed more abundant interstitial collagen deposition following degradation of the collagen threads. M2 macrophage supplementation improved structural and mechanical properties at the tissue and cellular level as indicated by increased modulus and stiffness. This study demonstrates improved biomechanical and histological outcomes following incorporation of M2 macrophages into genipin-cross-linked collagen biotextiles for tissue repair and offers future strategies focused on connective tissue regeneration. Impact statement Macrophages exhibit significant plasticity with complex phenotypes ranging from proinflammatory (M1) to proregenerative (M2). They release cytokines and chemokines governing immunological stability, inflammation resolution, and tissue healing and regeneration. However, utilization of macrophages as therapeutic tools for tissue engineering remains limited. In this study, genipin-cross-linked collagen biotextiles were employed to deliver M0, M1, and M2 macrophages and evaluate tissue responses and postsurgical mechanical properties in vivo. M2-seeded collagen biotextiles showed reduced fibrous capsule and favorable healing response. These outcomes shed new light on designing tissue-engineered constructs that offer a novel cell-based therapeutic approach for applications requiring structural augmentation.

The High-cycle Fatigue Life of Cortical Bone Allografts Is Radiation Sterilization Dose-dependent: An In Vitro Study.

BACKGROUND: Structural cortical bone allografts are a reasonable treatment option for patients with large cortical bone defects caused by trauma, tumors, or complications of arthroplasty. Although structural cortical bone allografts provide the benefit of an osteoconductive material, they are susceptible to fatigue failure (fracture) and carry a risk of disease transmission. Radiation-sterilization at the recommended dose of 25 kGy decreases the risk of disease transmission. However, previous studies demonstrated that radiation sterilization at this dose can negatively impact the high cycle-fatigue life of cortical bone. Although the effects of higher doses of radiation on cortical bone allografts are well described, the effects of lower doses of radiation on a high-cycle fatigue life of cortical bone are poorly understood. QUESTIONS/PURPOSES: (1) Does the cycle-fatigue life of human cortical allograft bone vary with gamma radiation dose levels of 0 (control), 10 kGy, 17.5 kGy, and 25 kGy? (2) What differences in Raman spectral biomarkers are observed following varying doses of gamma radiation exposure? METHODS: The high-cycle fatigue behavior of human cortical bone specimens was examined at different radiation sterilization doses under physiologic stress levels (35 MPa) and in a 37° C phosphate-buffered saline bath using a custom-designed rotating-bending fatigue device. Six human femora from three donors were obtained for this study (two male, 63 and 61 years old, respectively, and one female, 48 years old). Test specimens were allocated among four treatment groups (0 kGy [control], 10 kGy, 17.5 kGy, and 25 kGy) based on donor and anatomic location of harvest site (both length and cross-sectional quadrant of femoral diaphysis) to ensure equal variation (n = 13 per group). Specimens underwent high-cycle fatigue testing to failure. The number of cycles to failure was recorded. Raman spectroscopy (a noninvasive vibrational spectroscopy used to qualitatively assess bone quality) was used to detect whether any changes in Raman spectral biomarkers occurred after varying doses of gamma radiation exposure. RESULTS: There was a decrease in the log-transformed mean high-cycle fatigue life in specimens irradiated at 25 kGy (5.39 ± 0.32) compared with all other groups (0 kGy: 6.20 ± 0.50; 10k Gy: 6.35 ± 0.79; 17.5 kGy: 6.01 ± 0.53; p = 0.001). Specimens irradiated at 25 kGy were also more likely to exhibit a more brittle fracture surface pattern than specimens with more ductile fracture surface patterns irradiated at 0 kGy, 10 kGy, and 17.5 kGy (p = 0.04). The Raman biomarker for the ratio of the relative amount of disordered collagen to ordered collagen showed a decrease at the 10 kGy radiation level from 1.522 ± 0.025 preirradiation to 1.489 ± 0.024 postirradiation (p = 0.01); no other detectable changes in Raman biomarkers were observed. CONCLUSION: The high-cycle fatigue life of cortical bone undergoes a nonlinear, dose-dependent decrease with an increase in gamma radiation sterilization in a clinically relevant dose range (0-25 kGy). Importantly, a notable drop-off in the high-cycle fatigue life of cortical bone appeared to occur between 17.5 kGy and 25 kGy, correlating to a sixfold decrease in mean cycles to failure. We speculate that the decrease in the Raman biomarker for disordered collagen at 10 kGy with no loss in high-cycle fatigue life may be caused by an increased amount of nonenzymatic crosslinking of the collagen backbone relative to collagen chain-scission (whereas the benefits of crosslinking may be outweighed by excess scission of the collagen backbone at higher radiation doses), but future studies will need to ascertain whether this in fact is the case. CLINICAL RELEVANCE: Radiation sterilization at the industry standard of 25 kGy has a substantial negative impact on the high-cycle fatigue life of cortical bone. Given these findings, it is possible to provide a meaningful increase in the high-cycle fatigue life and improve the overall functional lifetime of cortical bone allografts by lowering the radiation-sterilization dose below 25 kGy. Future work on radiation-sterilization methods at these clinically relevant doses is warranted to aid in preserving the high cycle fatigue life of cortical bone allografts while maintaining sterility.

Staphylococcus aureus and Acinetobacter baumannii Inhibit Osseointegration of Orthopedic Implants.

Bacterial infections routinely cause inflammation and thereby impair osseointegration of orthopedic implants. Acinetobacter spp., which cause osteomyelitis following trauma, on or off the battlefield, were, however, reported to cause neither osteomyelitis nor osteolysis in rodents. We therefore compared the effects of Acinetobacter strain M2 to those of Staphylococcus aureus in a murine implant infection model. Sterile implants and implants with adherent bacteria were inserted in the femur of mice. Bacterial burden, levels of proinflammatory cytokines, and osseointegration were measured. All infections were localized to the implant site. Infection with either S. aureus or Acinetobacter strain M2 increased the levels of proinflammatory cytokines and the chemokine CCL2 in the surrounding femurs, inhibited bone formation around the implant, and caused loss of the surrounding cortical bone, leading to decreases in both histomorphometric and biomechanical measures of osseointegration. Genetic deletion of TLR2 and TLR4 from the mice partially reduced the effects of Acinetobacter strain M2 on osseointegration but did not alter the effects of S. aureus. This is the first report that Acinetobacter spp. impair osseointegration of orthopedic implants in mice, and the murine model developed for this study will be useful for future efforts to clarify the mechanism of implant failure due to Acinetobacter spp. and to assess novel diagnostic tools or therapeutic agents.

Raman spectroscopy-based water measurements identify the origin of MRI T2 signal in human articular cartilage zones and predict histopathologic score.

We investigated for the first time zonal-dependent water distribution in articular cartilage by Raman spectroscopy (RS). We further investigated the association of histopathologic score with RS- and magnetic resonance imaging (MRI)-based water measurements. Cadaveric human cartilage plugs (N = 16) with different osteoarthritis (OA) severity were used. Water content distribution in cartilage zones was probed using RS- and MRI-based techniques. Histopathologic scoring was performed by two independent observers blindly. Moderate associations existed between RS- and MRI-based water measurements across all cartilage zones. RS-based analysis of different water compartments helped assign the origin of the T2 signal collected from the various cartilage zones. RS-based water parameters significantly correlated with OA-severity score, whereas MRI-based water measurements did not. RS can probe different water compartments in cartilage zones and predict up to 66% of the variation observed in the histopathologic score. RS-based water measurement could be developed further to assess cartilage quality in the clinic.

Genipin guides and sustains the polarization of macrophages to the pro-regenerative M2 subtype via activation of the pSTAT6-PPAR-gamma pathway.

M2 macrophages are associated with deposition of interstitial collagen and other extracellular matrix proteins during the course wound healing and also inflammatory response to biomaterials. Developing advanced biomaterials to promote the M2 subtype may be an effective way to improve tissue reinforcement surgery outcomes. In this study, the effect of genipin, a naturally derived crosslinking agent, on M0 â†’ M2-polarization was investigated. Genipin was introduced either indirectly by seeding cells on aligned collagen biotextiles that are crosslinked by the agent or in soluble form by direct addition to the culture medium. Cellular elongation effects on macrophage polarization induced by the collagen biotextile were also investigated as a potential inducer of macrophage polarization. M0 and M2 macrophages demonstrated significant elongation on the surface of aligned collagen threads, while cells of the M1 subtype-maintained a round phenotype. M0 â†’ M2 polarization, as reflected by arginase and Ym-1 production, was observed on collagen threads only when the threads were crosslinked by genipin, implicating genipin as a more potent inducer of the regenerative phenotype compared to cytoskeletal elongation. The addition of genipin to the culture medium directly also drove the emergence of pro-regenerative phenotype as measured by the markers (arginase and Ym-1) and through the activation of the pSTAT6-PPAR-gamma pathway. This study indicates that genipin-crosslinked collagen biotextiles can be used as a delivery platform to promote regenerative response after biomaterial implantation. STATEMENT OF SIGNIFICANCE: The immune response is one of the key determinants of tissue repair and regeneration rate, and outcome. The M2 macrophage subtype is known to resolve the inflammatory response and support tissue repair by producing pro-regenerative factors. Therefore, a biomaterial that promotes M2 sub-type can be a viable strategy to enhance tissue regeneration. In this study, we investigated genipin-crosslinked electrochemically aligned collagen biotextiles for their capacity to induce pro-regenerative polarization of M0 macrophages. The results demonstrated that genipin, rather than matrix-induced cellular elongation, was responsible for M0 â†’ M2 polarization in the absence of other bioinductive factors and maintaining the M2 polarized status of macrophages. Furthermore, we identified that genipin polarizes the M2 macrophage phenotype via activation of the pSTAT6-PPAR-gamma pathway.

Chondrogenesis of Mesenchymal Stem Cells through Local Release of TGF-ß3 from Heparinized Collagen Biofabric.

Osteoarthritic degeneration of cartilage is a major social health problem. Tissue engineering of cartilage using combinations of scaffold and mesenchymal stem cells (MSCs) is emerging as an alternative to existing treatment options such as microfracture, mosaicplasty, allograft, autologous chondrocyte implantation, or total joint replacement. Induction of chondrogenesis in high-density pellets of MSCs is generally attained by soluble exogenous TGF-ß3 in culture media, which requires lengthy in vitro culture period during which pellets gain mechanical robustness. On the other hand, a growth factor delivering and a mechanically robust scaffold material that can accommodate chondroid pellets would enable rapid deployment of pellets after seeding. Delivery of the growth factor from the scaffold locally would drive the induction of chondrogenic differentiation in the postimplantation period. Therefore, we sought to develop a biomaterial formulation that will induce chondrogenesis in situ, and compared its performance to soluble delivery in vitro. In this vein, a heparin-conjugated mechanically robust collagen fabric was developed for sustained delivery of TGF-ß3. The amount of conjugated heparin was varied to enhance the amount of TGF-ß3 uptake and release from the scaffold. The results showed that the scaffold delivered TGF-ß3 for up to 8 days of culture, which resulted in 15-fold increase in GAG production, and six-fold increase in collagen synthesis with respect to the No TGF-ß3 group. The resulting matrix was cartilage like, in that type II collagen and aggrecan were positive in the spheroids. Enhanced chondrogenesis under in situ TGF-ß3 administration resulted in a Young's modulus of ∼600 kPa. In most metrics, there were no significant differences between the soluble delivery group and in situ heparin-mediated delivery group. In conclusion, heparin-conjugated collagen scaffold developed in this study guides chondrogenic differentiation of hMSCs in a mechanically competent tissue construct, which showed potential to be used for cartilage tissue regeneration. Impact statement The most significant finding of this study was that sustained release of TGF-ß3 from heparinized collagen scaffold had chondroinductive effect on pelleted human mesenchymal stem cells (hMSCs). The effect was comparable to that observed in hMSC pellets that were cultured in chondrogenic media supplemented with TGF-ß3. The stiffness of scaffolds at the baseline was about 50% that of native cartilage and over 28 days the combined stiffness of pellet/scaffold complex converged to the stiffness of native cartilage. These data indicate that the scaffold system can generate a load-bearing cartilage-like tissue by using hMSCs pellets in a mechanically competent framework.

Heparin-mediated antibiotic delivery from an electrochemically-aligned collagen sheet.

BACKGROUND: Implantable medical devices and hardware are prolific in medicine, but hardware associated infections remain a major issue. OBJECTIVE: To develop and evaluate a novel, biologic antimicrobial coating for medical implants. METHODS: Electrochemically compacted collagen sheets with and without crosslinked heparin were synthesized per a protocol developed by our group. Sheets were incubated in antibiotic solution (gentamicin or moxifloxacin) overnight, and in vitro activity was assessed with five-day diffusion assays against Pseudomonas aeruginosa. Antibiotic release over time from gentamicin-infused sheets was determined using in vitro elution and high performance liquid chromatography (HPLC). RESULTS: Collagen-heparin-antibiotic sheets demonstrated larger growth inhibition zones against P. aeruginosa compared to collagen-antibiotic alone sheets. This activity persisted for five days and was not impacted by rinsing sheets prior to evaluation. Rinsed collagen-antibiotic sheets did not produce any inhibition zones. Elution of gentamicin from collagen-heparin-gentamicin sheets was gradual and remained above the minimal inhibitory concentration for gentamicin-sensitive organisms for 29 days. Conversely, collagen-gentamicin sheets eluted their antibiotic load within 24 hours. Overall, heparin-associated sheets demonstrated larger inhibition zones against P. aeruginosa and prolonged elution profile via HPLC. CONCLUSION: We developed a novel, local antibiotic delivery system that could be used to coat medical implants/hardware in the future and reduce post-operative infections.