Anthony W. Behn

The effects of interactive mechanical and biochemical niche signaling on osteogenic differentiation of adipose-derived stem cells using combinatorial hydrogels.

Stem cells reside in a multi-factorial environment containing biochemical and mechanical signals. Changing biochemical signals in most scaffolds often leads to simultaneous changes in mechanical properties, which makes it difficult to elucidate the complex interplay between niche cues. Combinatorial studies on cell-material interactions have emerged as a tool to facilitate analyses of stem cell responses to various niche cues, but most studies to date have been performed on two-dimensional environments. Here we developed three-dimensional combinatorial hydrogels with independent control of biochemical and mechanical properties to facilitate analysis of interactive biochemical and mechanical signaling on adipose-derived stem cell osteogenesis in three dimensions. Our results suggest that scaffold biochemical and mechanical signals synergize only at specific combinations to promote bone differentiation. Leading compositions were identified to have intermediate stiffness (∼55kPa) and low concentration of fibronectin (10μg ml(-1)), which led to an increase in osteocalcin gene expression of over 130-fold. Our results suggest that scaffolds with independently tunable niche cues could provide a powerful tool for conducting mechanistic studies to decipher how complex niche cues regulate stem cell fate in three dimensions, and facilitate rapid identification of optimal niche cues that promote desirable cellular processes or tissue regeneration.

The Effect of Suture Coated With Mesenchymal Stem Cells and Bioactive Substrate on Tendon Repair Strength in a Rat Model

PURPOSE Exogenously administered mesenchymal stem cells and bioactive molecules are known to enhance tendon healing. Biomolecules have been successfully delivered using sutures that elute growth factors over time. We sought to evaluate the histologic and biomechanical effect of delivering both cells and bioactive substrates on a suture delivery vehicle in comparison with sutures coated with bioactive substrates alone. METHODS Bone marrow-derived stem cells were harvested from Sprague-Dawley rat femurs. Experimental cell and substrate-coated, coated suture (CS) group sutures were precoated with intercellular cell adhesion molecule 1 and poly-L-lysine and seeded with labeled bone marrow-derived stem cells. Control (substrate-only [SO] coated) group sutures were coated with intercellular cell adhesion molecule 1 and poly-L-lysine only. Using a matched-paired design, bilateral Sprague-Dawley rat Achilles tendons (n = 105 rats) were transected and randomized to CS or SO repairs. Tendons were harvested at 4, 7, 10, 14, and 28 days and subjected to histologic and mechanical assessment. RESULTS Labeled cells were present at repair sites at all time points. The CS suture repairs displayed statistically greater strength compared to SO repairs at 7 days (12.6 ± 5.0 N vs 8.6 ± 3.7 N, respectively) and 10 days (21.2 ± 4.9 N vs 16.4 ± 4.8 N, respectively). There was no significant difference between the strength of CS suture repairs compared with SO repairs at 4 days (8.1 ± 5.1 N vs 6.6 ± 2.3 N, respectively), 14 days (22.8 ± 7.3 N vs 25.1 ± 9.7 N, respectively), and 28 days (40.9 ± 12.4 N vs 34.6 ± 15.0 N, respectively). CONCLUSIONS Bioactive CS sutures enhanced repair strength at 7 to 10 days. There was no significant effect at later stages. CLINICAL RELEVANCE The strength nadir of a tendon repair occurs in the first 2 weeks after surgery. Bioactive suture repair might provide a clinical advantage by jump-starting the repair process during this strength nadir. Improved early strength might, in turn allow earlier unprotected mobilization.

Human flexor tendon tissue engineering: in vivo effects of stem cell reseeding.

Background: Tissue-engineered human flexor tendons may be an option to aid in reconstruction of complex upper extremity injuries with significant tendon loss. The authors hypothesize that human adipose-derived stem cells remain viable following reseeding on human tendon scaffolds in vivo and aid in graft integration. Methods: Decellularized human flexor tendons harvested from fresh-frozen cadavers and reseeded with green fluorescent protein–labeled pooled human adipose-derived stem cells were examined with bioluminescent imaging and immunohistochemistry. Reseeded repaired tendons were compared biomechanically with unseeded controls following implantation in athymic rats at 2 and 4 weeks. The ratio of collagen I to collagen III at the repair site was examined using Sirius red staining. To confirm cell migration, reseeded and unseeded tendons were placed either in contact or with a 1-mm gap for 12 days. Green fluorescent protein signal was then detected. Results: Following reseeding, viable cells were visualized at 12 days in vitro and 4 weeks in vivo. Biomechanical testing revealed no significant difference in ultimate load to failure and 2-mm gap force. Histologic evaluation showed host cell invasion and proliferation of the repair sites. No increase in collagen III was noted in reseeded constructs. Cell migration was confirmed from reseeded constructs to unseeded tendon scaffolds with tendon contact. Conclusions: Human adipose-derived stem cells reseeded onto decellularized allograft scaffolds are viable over 4 weeks in vivo. The movement of host cells into the scaffold and movement of adipose-derived stem cells along and into the scaffold suggests biointegration of the allograft.

Ankle Joint Contact Loads and Displacement With Progressive Syndesmotic Injury.

Background: Ligamentous injuries to the distal tibiofibular syndesmosis are predictive of long-term ankle dysfunction. Mild and moderate syndesmotic injuries are difficult to stratify, and the impact of syndesmosis injury on the magnitude and distribution of forces within the ankle joint during athletic activities is unknown. Methods: Eight below-knee cadaveric specimens were tested in the intact state and after sequential sectioning of the following ligaments: anterior-inferior tibiofibular, anterior deltoid (1 cm), interosseous/transverse (IOL/TL), posterior-inferior tibiofibular, and whole deltoid. In each condition, specimens were loaded in axial compression to 700 N and then externally rotated to 20 N·m torque. Results: During axial loading and external rotation, both the fibula and the talus rotated significantly after each ligament sectioning as compared to the intact condition. After IOL/TL release, a significant increase in posterior translation of the fibula was observed, although no syndesmotic widening was observed. Mean tibiotalar contact pressure increased significantly after IOL/TL release, and the center of pressure shifted posterolaterally, relative to more stable conditions, after IOL/TL release. There were significant increases in mean contact pressure and peak pressure along with a reduction in contact area with axial loading and external rotation as compared to axial loading alone for all 5 conditions. Conclusion: Significant increases in tibiotalar contact pressures occur when external rotation stresses are added to axial loading. Moderate and severe injuries are associated with a significant increase in mean contact pressure combined with a shift in the center of pressure and rotation of the fibula and talus. Clinical Relevance: Considerable changes in ankle joint kinematics and contact mechanics may explain why moderate syndesmosis injuries take longer to heal and are more likely to develop long-term dysfunction and, potentially, ankle arthritis.

Posterior Glenoid Wear in Total Shoulder Arthroplasty: Eccentric Anterior Reaming Is Superior to Posterior Augment

BackgroundUncorrected glenoid retroversion during total shoulder arthroplasty may lead to an increased likelihood of glenoid prosthetic loosening. Augmented glenoid components seek to correct retroversion to address posterior glenoid bone loss, but few biomechanical studies have evaluated their performance.Questions/purposesWe compared the use of augmented glenoid components with eccentric reaming with standard glenoid components in a posterior glenoid wear model. The primary outcome for biomechanical stability in this model was assessed by (1) implant edge displacement in superior and inferior edge loading at intervals up to 100,000 cycles, with secondary outcomes including (2) implant edge load during superior and inferior translation at intervals up to 100,000 cycles, and (3) incidence of glenoid fracture during implant preparation and after cyclic loading.MethodsA 12°-posterior glenoid defect was created in 12 composite scapulae, and the specimens were divided in two equal groups. In the posterior augment group, glenoid version was corrected to 8° and an 8°-augmented polyethylene glenoid component was placed. In the eccentric reaming group, anterior glenoid reaming was performed to neutral version and a standard polyethylene glenoid component was placed. Specimens were cyclically loaded in the superoinferior direction to 100,000 cycles. Superior and inferior glenoid edge displacements were recorded.ResultsSurviving specimens in the posterior augment group showed greater displacement than the eccentric reaming group of superior (1.01 ± 0.02 [95% CI, 0.89–1.13] versus 0.83 ± 0.10 [95% CI, 0.72–0.94 mm]; mean difference, 0.18 mm; p = 0.025) and inferior markers (1.36 ± 0.05 [95% CI, 1.24–1.48] versus 1.20 ± 0.09 [95% CI, 1.09–1.32 mm]; mean difference, 0.16 mm; p = 0.038) during superior edge loading and greater displacement of the superior marker during inferior edge loading (1.44 ± 0.06 [95% CI, 1.28–1.59] versus 1.16 ± 0.11 [95% CI, 1.02–1.30 mm]; mean difference, 0.28 mm; p = 0.009) at 100,000 cycles. No difference was seen with the inferior marker during inferior edge loading (0.93 ± 0.15 [95% CI, 0.56–1.29] versus 0.78 ± 0.06 [95% CI, 0.70–0.85 mm]; mean difference, 0.15 mm; p = 0.079). No differences in implant edge load were seen during superior and inferior loading. There were no instances of glenoid vault fracture in either group during implant preparation; however, a greater number of specimens in the eccentric reaming group were able to achieve the final 100,000 time without catastrophic fracture than those in the posterior augment group.ConclusionsWhen addressing posterior glenoid wear in surrogate scapula models, use of angle-backed augmented glenoid components results in accelerated implant loosening compared with neutral-version glenoid after eccentric reaming, as shown by increased implant edge displacement at analogous times.Clinical RelevanceAngle-backed components may introduce shear stress and potentially compromise stability. Additional in vitro and comparative long-term clinical followup studies are needed to further evaluate this component design.

Biomechanical Comparison of an All-Soft Suture Anchor With a Modified Broström-Gould Suture Repair for Lateral Ligament Reconstruction

Background: Anatomic repair is indicated for patients who have recurrent lateral ankle instability despite nonoperative measures. Hypothesis: There is no difference in repair stiffness, failure torque, or failure angle between specimens repaired with all-soft suture anchors versus the modified Broström-Gould technique with sutures only. Study Design: Controlled laboratory study. Methods: In 10 matched pairs of human cadaveric ankles, the anterior talofibular ligament (ATFL) was incised from its origin on the fibula. After randomization, 1 ankle was repaired to its anatomic insertion using two 1.4-mm JuggerKnot all-soft suture anchors; the other ankle was repaired with a modified Broström-Gould technique using 2-0 FiberWire. All were augmented using the inferior extensor retinaculum. All ankles were mounted to the testing machine in 20° of plantar flexion and 15° of internal rotation and loaded to failure after the repair. Stiffness, failure torque, and failure angle were recorded and compared using a paired Student t test with a significance level set at P < .05. Results: There was no significant difference in failure torque, failure angle, or stiffness. No anchors pulled out of bone. The primary mode of failure was pulling through the ATFL tissue. Conclusion: There was no statistical difference in strength or stiffness between a 1.4-mm all-soft suture anchor and a modified Broström-Gould repair with 2-0 FiberWire. The primary mode of failure was at the tissue level rather than knot failure or anchor pullout. Clinical Relevance: The particular implant choice (suture only, tunnel, anchor) in repairing the lateral ligament complex may not be as important as the time to biological healing. The suture-only construct as described in the Broström-Gould repair was as strong as all-soft suture anchors, and the majority of the ankles failed at the tissue level. For those surgeons whose preference is to use anchor repair, this novel all-soft suture anchor may be an alternative to other larger anchors, as none failed by pullout.

Physicochemical decellularization of composite flexor tendon-bone interface grafts.

Background: Extremity injuries involving tendon attachment to bone are difficult to address. Clinically, tendon-bone interface allografts must be decellularized to reduce immunogenicity. Composite grafts are difficult to decellularize because chemical agents cannot reach cells between tissues. In this study, the authors attempted to optimize tendon-bone interface graft decellularization. Methods: Human flexor digitorum profundus tendons with attached distal phalanx were harvested from cadavers and divided into four groups. Group 1 (control) was untreated. Group 2 (chemical) was chemically treated with 5% peracetic acid, 0.1% ethylenediaminetetraacetic acid, and 0.1% sodium dodecyl sulfate. Group 3 (low-power) underwent targeted ultrasonication for 3 minutes (22,274 J, 126W) followed by chemical decellularization. Group 4 (high-power) underwent targeted ultrasonication for 10 minutes (88,490 J, 155W) followed by chemical decellularization. Decellularization was assessed histologically with hematoxylin and eosin stain and stains for major histocompatibility complex I stains. Cell counts were performed. The ultimate tensile load of decellularized grafts (group 4) were compared with pair-matched untreated grafts (group 1). Results: Average cell counts were 100 ± 41, 27 ± 10, 12 ± 11, and 6 ± 11 per high-power field for groups 1, 2, 3, and 4, respectively (p < 0.001). Decellularization using physical and chemical treatments (groups 3 and 4) resulted in substantial reduction of cells and major histocompatibility complex I molecules. There was no difference in ultimate tensile load between treated (group4) and untreated (group 1) samples (p > 0.5). Conclusions: Physicochemical decellularization of tendon-bone interface grafts using targeted ultrasonication and chemical treatment resulted in near-complete reduction in cellularity and maintenance of tensile strength. In the future, these decellularized composite scaffolds may be used for reconstruction of tendon-bone injuries.

Comparative potential of juvenile and adult human articular chondrocytes for cartilage tissue formation in three-dimensional biomimetic hydrogels.

Regeneration of human articular cartilage is inherently limited and extensive efforts have focused on engineering the cartilage tissue. Various cellular sources have been studied for cartilage tissue engineering including adult chondrocytes, and embryonic or adult stem cells. Juvenile chondrocytes (from donors below 13 years of age) have recently been reported to be a promising cell source for cartilage regeneration. Previous studies have compared the potential of adult and juvenile chondrocytes or adult and osteoarthritic (OA) chondrocytes. To comprehensively characterize the comparative potential of young, old, and diseased chondrocytes, here we examined cartilage formation by juvenile, adult, and OA chondrocytes in three-dimensional (3D) biomimetic hydrogels composed of poly(ethylene glycol) and chondroitin sulfate. All three human articular chondrocytes were encapsulated in the 3D biomimetic hydrogels and cultured for 3 or 6 weeks to allow maturation and extracellular matrix formation. Outcomes were analyzed using quantitative gene expression, immunofluorescence staining, biochemical assays, and mechanical testing. After 3 and 6 weeks, juvenile chondrocytes showed a greater upregulation of chondrogenic gene expression than adult chondrocytes, while OA chondrocytes showed a downregulation. Aggrecan and type II collagen deposition and glycosaminoglycan accumulation were high for juvenile and adult chondrocytes but not for OA chondrocytes. Similar trend was observed in the compressive moduli of the cartilage constructs generated by the three different chondrocytes. In conclusion, the juvenile, adult and OA chondrocytes showed differential responses in the 3D biomimetic hydrogels. The 3D culture model described here may also provide a useful tool to further study the molecular differences among chondrocytes from different stages, which can help elucidate the mechanisms for age-related decline in the intrinsic capacity for cartilage repair.

The Effect of Donor Age on Structural and Mechanical Properties of Allograft Tendons

Background: Allograft tendons are commonly used in surgical ligament reconstruction. While it is commonly accepted that donor age will affect mechanical properties of graft tissue, the apparent age threshold is unknown. Hypothesis: Donor age will significantly influence the structural and mechanical properties of tibialis posterior allograft tendons. Study Design: Controlled laboratory study. Methods: A total of 550 allograft posterior tibialis tendons were examined. Linear stiffness, ultimate tensile force, ultimate displacement, tensile modulus, ultimate tensile strength, and ultimate tensile strain were calculated for specimens from donors in each of 6 age groups: 15-29, 30-39, 40-49, 50-59, 60-69, and 70-79 years. Both first- and second-order polynomial regressions were performed to determine the correlation between structural and mechanical properties and age. Welch analyses of variance with Games-Howell post hoc tests were performed to facilitate comparisons among age groups. Results: All parameters displayed a weak correlation with age, with the highest R 2 term being 0.063 for ultimate tensile strength. Linear stiffness, ultimate tensile force, and tensile modulus displayed almost no correlation with age. Ultimate tensile strength increased slightly with age up to 40-49 years and then decreased with further increases in age. Slight decreases in ultimate displacement and ultimate tensile strain were observed with increasing age. Numerous statistically significant differences were observed between age groups for each outcome parameter; however, the magnitudes of the differences between age groups are relatively small (<15%) for all outcome parameters. Conclusion: Age explained at most 6% of the variation in structural and mechanical properties of tibialis posterior allograft tendons. Clinical Relevance: Posterior tibialis tendons from all age groups displayed structural properties superior to the native anterior cruciate ligament, with higher stiffness and ultimate force, and less displacement to failure. Although statistically significant differences in structural and mechanical properties were observed between age groups, the magnitudes of the differences are small and most likely not clinically relevant. The age of the donor will not likely affect the suitability of a graft for use in surgical reconstruction.

The effect of local IL‐4 delivery or CCL2 blockade on implant fixation and bone structural properties in a mouse model of wear particle induced osteolysis

Modulation of macrophage polarization and prevention of CCL2-induced macrophage chemotaxis are emerging strategies to reduce wear particle induced osteolysis and aseptic total joint replacement loosening. In this study, the effect of continuous IL-4 delivery or bioactive implant coating that constitutively releases a protein inhibitor of CCL2 signaling (7ND) on particle induced osteolysis were studied in the murine continuous femoral intramedullary particle infusion model. Polyethylene particles with or without IL-4 were infused into mouse distal femurs implanted with hollow titanium rods using subcutaneous infusion pumps. In another experimental group, particles were infused into the femur through a 7ND coated rod. After 4 weeks, fixation of the implant was assessed using a pullout test. The volume of trabecular bone and the geometry of the local cortical bone were assessed by µCT and the corresponding structural properties of the cortical bone determined by torsional testing. Continuous IL-4 delivery led to increased trabecular bone volume as well as enhanced local bone geometry and structural properties, while 7ND implant coating did not have effect on these parameters. The results suggest that local IL-4 treatment is a promising strategy to mitigate wear particle induced osteolysis.