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.

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.

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.

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.

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.

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.

Diffuse microdamage in bone activates anabolic response by osteoblasts via involvement of voltage-gated calcium channels.

INTRODUCTION: Matrix damage sustained by bone tissue is repaired by the concerted action of bone cells. Previous studies have reported extracellular calcium ([Ca2+]E) efflux to originate from regions of bone undergoing diffuse microdamage termed as "diffuse microdamage-induced calcium efflux" (DMICE). DMICE has also been shown to activate and increase intracellular calcium ([Ca2+]I) signaling in osteoblasts via the involvement of voltage-gated calcium channels (VGCC). Past studies have assessed early stage (< 1 h) responses of osteoblasts to DMICE. The current study tested the hypothesis that DMICE has longer-term sustained effect such that it induces anabolic response of osteoblasts. MATERIALS AND METHODS: Osteoblasts derived from mouse calvariae were seeded on devitalized bovine bone wafers. Localized diffuse damage was induced in the vicinity of cells by bending. The response of osteoblasts to DMICE was evaluated by testing gene expression, protein synthesis and mineralized nodule formation. RESULTS: Cells on damaged bone wafers showed a significant increase in RUNX2 and Osterix expression compared to non-loaded control. Also, RUNX2 and Osterix expression were suppressed significantly when the cells were treated with bepridil, a non-selective VGCC inhibitor, prior to loading. Significantly higher amounts of osteocalcin and mineralized nodules were synthesized by osteoblasts on diffuse damaged bone wafers, while bepridil treatment resulted in a significant decrease in osteocalcin production and mineralized nodule formation. CONCLUSION: In conclusion, this study demonstrated that DMICE activates anabolic responses of osteoblasts through activation of VGCC. Future studies of osteoblast response to DMICE in vivo will help to clarify how bone cells repair diffuse microdamage.

Raman Biomarkers Are Associated with Cyclic Fatigue Life of Human Allograft Cortical Bone.

BACKGROUND: Structural bone allografts are an established treatment method for long-bone structural defects resulting from such conditions as traumatic injury and sarcoma. The functional lifetime of structural allografts depends on resistance to cyclic loading (cyclic fatigue life), which can lead to fracture at stress levels well below the yield strength. Raman spectroscopy biomarkers can be used to non-destructively assess the 3 primary components of bone (collagen, mineral, and water), and may aid in optimizing allograft selection to decrease fatigue fracture risk. We studied the association of Raman biomarkers with the cyclic fatigue life of human allograft cortical bone. METHODS: Twenty-one cortical bone specimens were machined from the femoral diaphyses of 4 human donors (a 63-year old man, a 61-year-old man, a 51-year-old woman, and a 48-year-old woman) obtained from the Musculoskeletal Transplant Foundation. Six Raman biomarkers were analyzed: collagen disorganization, mineral maturation, matrix mineralization, and 3 water compartments. The specimens underwent cyclic fatigue testing under fully reversed conditions (35 and 45 MPa), during which they were tested to fracture or to 30 million cycles ("runout"), simulating 15 years of moderate activity. A tobit censored linear regression model for cyclic fatigue life was created. RESULTS: The multivariate model explained 60% of the variance in the cyclic fatigue life (R = 0.604, p < 0.001). Increases in Raman biomarkers for disordered collagen (coefficient: -2.74×10, p < 0.001) and for loosely collagen-bound water compartments (coefficient: -2.11×10, p < 0.001) were associated with a decreased cyclic fatigue life. Increases in Raman biomarkers for mineral maturation (coefficient: 3.50×10, p < 0.001), matrix mineralization (coefficient: 2.32×10, p < 0.001), tightly collagen-bound water (coefficient: 1.19×10, p < 0.001), and mineral-bound water (coefficient: 3.27×10, p < 0.001) were associated with an increased cyclic fatigue life. Collagen disorder accounted for 44% of the variance in the cyclic fatigue life, mineral maturation accounted for 6%, and all bound water compartments accounted for 3%. CONCLUSIONS: Increasing baseline collagen disorder was associated with a decreased cyclic fatigue life and had the strongest correlation with the cyclic fatigue life of human cortical donor bone. This model should be prospectively validated. CLINICAL RELEVANCE: Raman analysis is a promising tool for the non-destructive evaluation of structural bone allograft quality for load-bearing applications.

Microbially-derived nanofibrous cellulose polymer for connective tissue regeneration.

Among a vast array of biomaterials investigated for tissue engineering applications, bacterial cellulose (BC) has not been evaluated in depth, despite the material's strong potential of applicability in the field of biotechnology. In this study we investigate the effect of sugar concentration and culture duration on physical and mechanical properties of BC. BC was grown in culture media with different glucose concentrations (weight percent) of 1.25%, 2.50%, 5.00%, 10.00%, 15.00% and also in media with fructose concentration of 5.00%. The swelling ratio of harvested BC sheets did not change significantly with concentration of glucose or the type of sugar (fructose vs glucose). Swelling ratio did not change significantly with culture duration either. Cellulose production rate was significantly higher (p < 0.05) at 5.00%wt. glucose concentration compared to other groups. Ultimate tensile strength (309.3 ±â€¯32.8 MPa) and Young's modulus (3.1 ±â€¯0.6 GPa) of BC sheets harvested from the medium with 5.00%wt. glucose concentration were the highest among all treatment groups. Bacterial removal process and testing condition (wet/dry) did not affect the mechanical performance of the bacterial cellulose significantly. X-ray diffraction data demonstrated higher crystallinity for samples cultured in media with 5.00%wt. glucose concentration. Viability/cytotoxicity, proliferation, and cells' metabolic activities demonstrated BC to be biocompatible. Cells attached, spread, and proliferated with time on bacterial cellulose. Results of this study showed 5.00 wt% glucose concentration is the optimum concentration of sugar in media to produce BC with highest strength and modulus compared to other concentration. High mechanical strength along with biocompatibility present bacterial cellulose as an invaluable material for use in tissue engineering of load bearing connective tissues such as tendons and ligaments.

Woven collagen biotextiles enable mechanically functional rotator cuff tendon regeneration during repair of segmental tendon defects in vivo.

Despite advancements in surgical techniques and materials for rotator cuff repair procedures, primary repair failures remain common. This study examines the use of electrochemically aligned collagen (ELAC) threads woven into biotextile scaffolds as grafts to repair critical infraspinatus tendon defects in New Zealand White rabbits. Three surgical treatment groups were evaluated: rabbits undergoing direct repair as operative controls, rabbits receiving ELAC scaffolds alone, and rabbits treated with mesenchymal stem cell (MSC)-seeded ELAC scaffolds. In each animal, the intact, contralateral infraspinatus served as an internal positive control. Tendon-bone constructs were harvested after 3 months in vivo and outcome measures included biomechanical testing, histological staining, and immunohistochemical staining. Biomechanical testing revealed that maximum load-bearing capacity was comparable between all groups, while MSC-seeded scaffold repairs exhibited increased stiffness relative to non-seeded scaffold repairs. Histological staining revealed robust collagen deposition around ELAC fibers and increased cellularity within the continuum of woven scaffolds as compared to native tendon. Immunohistochemical staining revealed presence of collagens I and III in all groups, but procollagen I and the tendon-specific marker tenomodulin were only observed in seeded and non-seeded ELAC scaffold repairs. Findings of this pilot study warrant continued investigation of ELAC biotextile scaffolds for repair of critically-sized rotator cuff tendon defects. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B: 1864-1876, 2019.

In vivo biocompatibility and time-dependent changes in mechanical properties of woven collagen meshes: A comparison to xenograft and synthetic mid-urethral sling materials.

Meshes woven from highly aligned collagen threads crosslinked using either genipin or 1-ethyl-3-(3-dimethylaminopropyl) carboiimide and N-hydroxy succinimide (EDC/NHS) were implanted in a subcutaneous rat model to evaluate their biocompatibility (at 2 weeks, 2 months, and 5 months), mechanical properties (at baseline, 2 months, and 5 months) and ultimately their suitability for use as mid-urethral slings (MUS) for management of stress urinary incontinence. Porcine dermal (Xenmatrix) and monofilament polypropylene (Prolene) meshes were also implanted to provide comparison to clinically used materials. Quantitative histological scoring showed tissue integration in Xenmatrix was almost absent, while the open network of woven collagen and Prolene meshes allowed for cellular and tissue integration. However, strength and stiffness of genipin-crosslinked collagen (GCC), Prolene, and Xenmatrix meshes were not significantly different from those of native rectus fascia and vaginal tissues of animals at 5 months. EDC/NHS-crosslinked collagen (ECC) meshes were degraded so extensively at five months that samples could only be used for histological staining. Picrosirius red and Masson's trichrome staining revealed that integrated tissue within GCC meshes was more aligned (p = 0.02) and appeared more concentrated than ECC meshes at 5 months. Furthermore, immunohistochemical staining showed that GCC meshes attracted a greater number of cells expressing markers for M2 macrophages, those associated with regeneration, than ECC meshes (p = 0.01 for CD206+ cells, p = 0.001 CD163+ cells) at 5 months. As such, GCC meshes hold promise as a new MUS biomaterial based on favorable induction of fibrous tissue resulting in mechanical stiffness matching that of native tissue. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 2018. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B: 479-489, 2019.

Kinesin and Dynein Mechanics: Measurement Methods and Research Applications.

Motor proteins play critical roles in the normal function of cells and proper development of organisms. Among motor proteins, failings in the normal function of two types of proteins, kinesin and dynein, have been shown to lead many pathologies, including neurodegenerative diseases and cancers. As such, it is critical to researchers to understand the underlying mechanics and behaviors of these proteins, not only to shed light on how failures may lead to disease, but also to guide research toward novel treatment and nano-engineering solutions. To this end, many experimental techniques have been developed to measure the force and motility capabilities of these proteins. This review will (a) discuss such techniques, specifically microscopy, atomic force microscopy (AFM), optical trapping, and magnetic tweezers, and (b) the resulting nanomechanical properties of motor protein functions such as stalling force, velocity, and dependence on adenosine triphosophate (ATP) concentrations will be comparatively discussed. Additionally, this review will highlight the clinical importance of these proteins. Furthermore, as the understanding of the structure and function of motor proteins improves, novel applications are emerging in the field. Specifically, researchers have begun to modify the structure of existing proteins, thereby engineering novel elements to alter and improve native motor protein function, or even allow the motor proteins to perform entirely new tasks as parts of nanomachines. Kinesin and dynein are vital elements for the proper function of cells. While many exciting experiments have shed light on their function, mechanics, and applications, additional research is needed to completely understand their behavior.

Effects of PDGF-BB delivery from heparinized collagen sutures on the healing of lacerated chicken flexor tendon in vivo.

Flexor tendon lacerations are traditionally repaired by using non-absorbable monofilament sutures. Recent investigations have explored to improve the healing process by growth factor delivery from the sutures. However, it is difficult to conjugate growth factors to nylon or other synthetic sutures. This study explores the performance of a novel electrochemically aligned collagen suture in a flexor tendon repair model with and without platelet derived growth factor following complete tendon laceration in vivo. Collagen suture was fabricated via electrochemical alignment process. Heparin was covalently bound to electrochemically aligned collagen sutures (ELAS) to facilitate affinity bound delivery of platelet-derived growth factor-BB (PDGF-BB). Complete laceration of the flexor digitorum profundus in the third digit of the foot was performed in 36 skeletally mature White Leghorn chickens. The left foot was used as the positive control. Animals were randomly divided into three groups: control specimens treated with standard nylon suture (n=12), specimens repaired with heparinated ELAS suture without PDGF-BB (n=12) and specimens repaired with heparinated ELAS suture with affinity bound PDGF-BB (n=12). Specimens were harvested at either 4weeks or 12weeks following tendon repair. Differences between groups were evaluated by the degree of gross tendon excursion, failure load/stress, stiffness/modulus, absorbed energy at failure, elongation/strain at failure. Quantitative histological scoring was performed to assess cellularity and vascularity. Closed flexion angle measurements demonstrated no significant differences in tendon excursion between the study groups at 4 or 12weeks. Biomechanical testing showed that the group treated with PDGF-BB bound heparinated ELAS suture had significantly higher stiffness and failure load (p<0.05) at 12-weeks relative to both heparinated ELAS suture and nylon suture. Similarly, the group treated with PDGF-BB bound suture had significantly higher ultimate tensile strength and Young's modulus (p<0.05) at 12-weeks relative to both ELAS suture and nylon suture. Compared to nylon controls, heparinized ELAS with PDGF-BB improved biomechanics and vascularity during tendon healing by 12-weeks following primary repair. The ability of ELAS to deliver PDGF-BB to the lacerated area of tendon presents investigators with a functional bioinductive platform to improve repair outcomes following flexor tendon repair. STATEMENT OF SIGNIFICANCE: A high strength aligned collagen suture was fabricated via linear electrocompaction and heparinized for prolonged delivery of PDFG-BB. When it was used to suture a complete lacerated flexor tendon in a chicken model controlled release of the PDGF-BB improved the strength of treated tendon after 12 weeks compared to tendon sutured with commercial nylon suture. Furthermore, Collagen suture with affinity bound PDGF-BB enhanced the vascularization and remodeling of lacerated tendon when it compare to synthetic nylon suture. Overall, electrocompacted collagen sutures holds potential to improve repair outcome in flexor tendon surgeries by improving repair strength and stiffness, vascularity, and remodeling via sustained delivery of the PDGF-BB. The bioinductive collagen suture introduces a platform for sustained delivery of other growth factors for a wide-array of applications.

Raman spectral markers of collagen denaturation and hydration in human cortical bone tissue are affected by radiation sterilization and high cycle fatigue damage.

BACKGROUND: Thermal denaturation and monotonic mechanical damage alter the organic and water-related compartments of cortical bone. These changes can be detected using Raman spectroscopy. However, less is known regarding Raman sensitivity to detect the effects of cyclic fatigue damage and allograft sterilization doses of gamma radiation. OBJECTIVE: To determine if Raman spectroscopic biomarkers of collagen denaturation and hydration are sensitive to the effects of (a) high cycle fatigue damage and (b) 25kGy irradiation. METHODS: Unirradiated and gamma-radiation sterilized human cortical bone specimens previously tested in vitro under high-cycle (> 100,000 cycles) fatigue conditions at 15MPa, 25MPa, 35MPa, 45MPa, and 55MPa cyclic stress levels were studied. Cortical bone Raman spectral profiles from wavenumber ranges of 800-1750cm-1 and 2700-3800cm-1 were obtained and compared from: a) non-fatigue vs fatigue fracture sites and b) radiated vs. unirradiated states. Raman biomarker ratios 1670/1640 and 3220/2949, which reflect collagen denaturation and organic matrix (mainly collagen)-bound water, respectively, were assessed. One- and two-way ANOVA analyses were utilized to identify differences between groups along with interaction effects between cyclic fatigue and radiation-induced damage. RESULTS: Cyclic fatigue damage resulted in increases in collagen denaturation (1670/1640: 1.517 ± 0.043 vs 1.579 ± 0.021, p < 0.001) and organic matrix-bound water (3220/2949: 0.109 ± 0.012 vs 0.131 ± 0.008, p < 0.001). Organic matrix-bound water increased secondary to 25kGy irradiation (3220/2949: 0.105 ± 0.010 vs 0.1161 ± 0.009, p = 0.003). Organic matrix-bound water was correlated positively with collagen denaturation (r = 0.514, p < 0.001). CONCLUSIONS: Raman spectroscopy can detect the effects of cyclic fatigue damage and 25kGy irradiation via increases in organic matrix (mainly collagen)-bound water. A Raman measure of collagen denaturation was sensitive to cyclic fatigue damage but not 25kGy irradiation. Collagen denaturation was correlated with organic matrix-bound water, suggesting that denaturation of collagen to gelatinous form may expose more binding sites to water by unwinding the triple alpha chains. This research may eventually be useful to help identify allograft quality and more appropriately match donors to recipients.

Femoral Iatrogenic Subtrochanteric Fatigue Fracture Risk is not Increased by Placing Drill Holes Below the Level of the Lesser Trochanter.

BACKGROUND: Iatrogenic subtrochanteric fractures of the femur can occur postoperatively following placement of screws in the lateral femoral cortex. Drilling holes below the lesser trochanter is generally avoided to prevent fatigue failure; however, there is little biomechanical evidence to support this recommendation. We hypothesized that hole placement below the level of the lesser trochanter will not accelerate fatigue failure compared to holes at the level of the lesser trochanter. METHODS: Twelve matched-pairs of male fresh-frozen cadaveric femurs were used for biomechanical testing. A single screw hole was drilled through the lateral femoral cortex either at the level of the lesser trochanter (proximal-hole group) or below the lesser trochanter (distal-hole group). Each femur was cycled to failure using a physiologically-relevant loading model. Paired t-test was used to evaluate for a difference in cycles to failure between groups. RESULTS: There was no statistical difference in cycles to failure between the groups with the hole drilled at or below the lesser trochanter. CONCLUSIONS: The traditional recommendation to avoid drilling holes below the level of the lesser trochanter is based mainly on experience and case reports in the literature. The results of this study indicate that placing holes below the level of the lesser trochanter, in and of itself, may not pose any additional risk of fracture. Other important factors need to be considered, such as tapering of the lateral femoral cortex. CLINICAL RELEVANCE: There are often situations where the patient's anatomy and facture pattern is more conducive to placing a screw distal to the lesser trochanter. This study may allow surgeons greater flexibility in placing screws more distally in the lateral femoral cortex by demonstrating the safety of doing so, at least in the population studied.

Effects of substrate stiffness on the tenoinduction of human mesenchymal stem cells.

Extracellular matrix modulus plays an important role in regulating cell morphology, proliferation and differentiation during regular and diseased states. Although the effects of substrate topography and modulus on MSC differentiation are well known with respect to osteogenesis and adipogenesis, there has been relatively little investigation on the effects of this phenomenon on tenogenesis. Furthermore, relative roles of topographical factors (matrix alignment vs. matrix modulus) in inducing tenogenic differentiation is not well understood. In this study we investigated the effects of modulus and topographical alignment of type I collagen substrate on tendon differentiation. Type I collagen sheet substrates with random topographical alignment were fabricated with their moduli tuned in the range of 0.1, 1, 10 and 100MPa by using electrocompaction and controlled crosslinking. In one of the groups, topographical alignment was introduced at 10MPa stiffness, by controlled unidirectional stretching of the sheet. RT-PCR, immunohistochemistry and immunofluorescence results showed that mimicking the tendon topography, i.e. increasing the substrate modulus as well as alignment increased the tenogenic differentiation. Higher substrate modulus increased the expression of COLI, COLIII, COMP and TSP-4 about 2-3-fold and increased the production of COLI, COLIII and TSP-4 about 2-4-fold. Substrate alignment up regulated COLIII and COMP expression by 2-fold. Therefore, the tenoinductive collagen material model developed in this study can be used in the research and development of tissue engineering tendon repair constructs in future. STATEMENT OF SIGNIFICANCE: Although the effects of substrate topography and modulus on MSC differentiation are well known with respect to osteogenesis and adipogenesis, there has been relatively little investigation on the effects of this phenomenon on tenogenesis. Furthermore, a relative role of topographical factors (matrix alignment vs. matrix modulus) in inducing tenogenic differentiation is not well understood. We investigated the effects of modulus and topographical alignment of type I collagen substrate on tendon differentiation. This study showed mimicking the tendon topography, i.e. increasing the substrate modulus as well as alignment increased the tenogenic differentiation. Therefore, the tenoinductive collagen material model developed in this study can be used in the research and development of tissue engineering tendon repair constructs in future.