Macrophage depletion impairs neonatal tendon regeneration.

Tendons are dense connective tissues that transmit muscle forces to the skeleton. After adult injury, healing potential is generally poor and dominated by scar formation. Although the immune response is a key feature of healing, the specific immune cells and signals that drive tendon healing have not been fully defined. In particular, the immune regulators underlying tendon regeneration are almost completely unknown due to a paucity of tendon regeneration models. Using a mouse model of neonatal tendon regeneration, we screened for immune-related markers and identified upregulation of several genes associated with inflammation, macrophage chemotaxis, and TGFß signaling after injury. Depletion of macrophages using AP20187 treatment of MaFIA mice resulted in impaired functional healing, reduced cell proliferation, reduced ScxGFP+ neo-tendon formation, and altered tendon gene expression. Collectively, these results show that inflammation is a key component of neonatal tendon regeneration and demonstrate a requirement for macrophages in effective functional healing.

The Posterior Bundle's Effect on Posteromedial Elbow Instability After a Transverse Coronoid Fracture: A Biomechanical Study.

PURPOSE: There has been increased interest in the role of the posterior bundle of the medial collateral ligament (pMUCL) in the elbow, particularly its effects on posteromedial rotatory stability. The ligament's effect in the context of an unfixable coronoid fracture has not been the focus of any study. The purposes of this biomechanical study were to evaluate the stabilizing effect of the pMUCL with a transverse coronoid fracture and to assess the effect of graft reconstruction of the ligament. METHODS: We simulated a varus and internal rotatory subluxation in 7 cadaveric elbows at 30°, 60°, and 90° elbow flexion. The amount of ulnar rotation and medial ulnohumeral joint gapping were assessed in the intact elbow after we created a transverse coronoid injury, after we divided the pMUCL, and finally, after we performed a graft reconstruction of the pMUCL. RESULTS: At all angles tested, some stability was lost after cutting the pMUCL once the coronoid had been injured, because mean proximal ulnohumeral joint gapping increased afterward by 2.1, 2.2, and 1.3 mm at 90°, 60°, and 30°, respectively. Ulnar internal rotation significantly increased after pMUCL transection at 90°. At 60° and 30° elbow flexion, ulnar rotation increased after resection of the coronoid but not after pMUCL resection. CONCLUSIONS: An uninjured pMUCL stabilizes against varus internal rotatory instability in the setting of a transverse coronoid fracture at higher flexion angles. Further research is needed to optimize graft reconstruction of the pMUCL. CLINICAL RELEVANCE: The pMUCL is an important secondary stabilizer against posteromedial instability in the coronoid-deficient elbow. In the setting of an unfixable coronoid fracture, the surgeon should examine for posteromedial instability and consider addressing the pMUCL surgically.

Varus posteromedial rotatory instability: a biomechanical analysis of posterior bundle of the medial ulnar collateral ligament reconstruction.

BACKGROUND: Recently, there has been growing interest in the involvement of the posterior bundle of the medial ulnar collateral ligament (pMUCL) in varus posteromedial rotatory instability (PMRI). Varus PMRI has been observed clinically, but the degree of involvement of the pMUCL remains unclear. This study assessed the degree to which the pMUCL is involved in stabilizing the elbow and the feasibility of a pMUCL reconstruction to restore stability. METHODS: Movements simulating PMRI were performed in 8 cadaveric elbows. Joint gapping values were obtained by 3-dimensional motion capture for the proximal and distal aspects of the ulnohumeral joint. Specimens were assessed at "intact," "cut coronoid + pMUCL," "reconstruction," and "cut anterior aspect MUCL + reconstruction" conditions with mechanical testing at 30°, 60°, and 90° of elbow flexion. RESULTS: Proximal joint gapping significantly increased from intact to cut coronoid + pMUCL at 60° and 90°, and distal joint gapping significantly increased at 90°. In the reconstruction condition, joint gapping across the proximal joint at 60° and 90° significantly recovered, as did distal joint gapping at 90°. In the cut anterior aspect MUCL + reconstruction condition, no significant increase occurred in proximal or distal joint gapping. CONCLUSIONS: Transection of the pMUCL with a coronoid fracture leads to increased joint gapping, suggesting the presence of PMRI. PMRI can still occur with an intact lateral ligamentous complex. A pMUCL tendon graft reconstruction confers some elbow stability in this injury mechanism.

Structural and Chemical Modification to Improve Adhesive and Material Properties of Fibrin-Genipin for Repair of Annulus Fibrosus Defects in Intervertebral Disks.

Annulus fibrosus (AF) defects from intervertebral disk (IVD) herniation and degeneration are commonly associated with back pain. Genipin-crosslinked fibrin hydrogel (FibGen) is an injectable, space-filling AF sealant that was optimized to match AF shear properties and partially restored IVD biomechanics. This study aimed to enhance mechanical behaviors of FibGen to more closely match AF compressive, tensile, and shear properties by adjusting genipin crosslink density and by creating a composite formulation by adding Poly(D,L-lactide-co-glycolide) (PDLGA). This study also evaluated effects of thrombin concentration and injection technique on gelation kinetics and adhesive strength. Increasing FibGen genipin concentration from 1 to 36 mg/mL significantly increased adhesive strength (∼5 to 35 kPa), shear moduli (∼10 to 110 kPa), and compressive moduli (∼25 to 150 kPa) with concentration-dependent effects, and spanning native AF properties. Adding PDLGA to FibGen altered the material microstructure on electron microscopy and nearly tripled adhesive strength, but did not increase tensile moduli, which remained nearly 5× below native AF, and had a small increase in shear moduli and significantly decreased compressive moduli. Increased thrombin concentration decreased gelation rate to < 5 min and injection methods providing a structural FibGen cap increased pushout strength by ∼40%. We conclude that FibGen is highly modifiable with tunable mechanical properties that can be formulated to be compatible with human AF compressive and shear properties and gelation kinetics and injection techniques compatible with clinical discectomy procedures. However, further innovations, perhaps with more efficient fiber reinforcement, will be required to enable FibGen to match AF tensile properties.

Isolated ligamentous injury can cause posteromedial elbow instability: a cadaveric study.

BACKGROUND: Elbow posteromedial rotatory instability (PMRI) is known to occur with fracture of the anteromedial coronoid and injury to the posterior bundle of the medial ulnar collateral ligament (pMUCL). However, whether instability results from isolated pMUCL injury remains unclear. The purpose of this study was to quantify displacement about the ulnohumeral joint to evaluate whether isolated sectioning of the pMUCL results in elbow PMRI. METHODS: Nine cadaveric elbows underwent movements simulating PMRI by application of axial compression with varus and internal rotation moments. Gapping values at both the proximal and distal aspects of the medial ulnohumeral joint were then recorded for "intact" and "pMUCL-sectioned" elbows at positions of 30°, 60°, and 90° of flexion. RESULTS: After pMUCL transection, torsion increased by 2.6° ± 0.7° (P = .054) at 30° and 4.5° ± 1.2° (P = .039) at 60° of flexion. Proximal ulnohumeral joint gapping also increased at 30° (1.4 ± 0.4 mm; P = .039), 60° (1.5 ± 0.6 mm; P = .039), and 90° (1.5 ± 0.7 mm; P = .017), respectively. No increases in distal ulnohumeral gapping occurred at any angle of flexion. DISCUSSION: Sectioning of the pMUCL results in significant increases in torsion and displacement about the proximal ulnohumeral joint. Our findings demonstrate that elbow PMRI can occur secondary to isolated ligamentous injury. Clinicians mindful of this previously unrecognized role of the pMUCL as a stabilizer may wish to consider methods of restoring pMUCL integrity when treating medial elbow instability.

Importance of the posterior bundle of the medial ulnar collateral ligament.

BACKGROUND: There has been a renewed interest in the pathomechanics of elbow dislocation, with recent literature having suggested that the medial ulnar collateral ligament is more often disrupted in dislocations than the lateral ligamentous complex. The purpose of this serial sectioning study was to determine the influence of the posterior bundle of the medial ulnar collateral ligament (pMUCL) as a stabilizer against elbow dislocation. METHODS: An elbow dislocation was simulated in 5 cadaveric elbows by mechanically applying an external rotation moment and valgus force. Medial ulnohumeral joint gapping was measured at 30°, 60°, and 90° of flexion in an intact elbow after sectioning of the medial collateral ligament's anterior bundle (aMUCL) and then after sectioning of the pMUCL as well. RESULTS: After sectioning of the aMUCL, the pMUCL was able to stabilize the joint against dislocation. After aMUCL sectioning, the proximal joint space significantly increased by 4.2 ± 0.6 mm at 30° of flexion and 2.6 ± 0.3 mm at 60° of flexion, although it did not dislocate. The gapping increase of 0.9 ± 0.6 at 90° of flexion did not reach significance. After sectioning of the pMUCL (after having already sectioned the aMUCL), all of the specimens frankly dislocated at all flexion angles. CONCLUSIONS: An intact pMUCL can prevent elbow dislocation and limited joint subluxation to within 6.6 mm. Our findings indicate that repair or reconstruction may be warranted in certain circumstances (ie, residual instability after operative management of a terrible triad injury or after aMUCL reconstruction).

Orthogonal plating of distal femur fractures: A biomechanical comparison with plate-nail and parallel plating constructs.

Purpose: This study compared the biomechanical properties of orthogonal plating with plate-nail and parallel plating constructs for supracondylar distal femur fractures. Methods: A supracondylar distal femur fracture was simulated using 15 synthetic osteoporotic femurs. Constructs included: (1) plate-nail (lateral locked distal femoral plate + retrograde intramedullary nail); (2) parallel plating (lateral locked distal femoral plate + medial 4.0 mm compression plate); and (3) orthogonal plating (lateral locked distal femoral plate + posterior one-third tubular plate). Specimens underwent nondestructive loading, fatigue loading, and loading to failure. Gapping at the fracture was measured using a three-dimensional motion capture system. Baseline torsional and axial stiffness, stiffness and strain after fatigue loading, and load to failure were determined. A case example of orthogonal plating is also presented. Results: There was no difference in baseline torsional (p = 0.51) and axial stiffness (p = 0.53). Stiffness after fatigue loading was highest with parallel plating, with no difference between the plate-nail and orthogonal plating constructs (p = 0.84). Strain after fatigue loading was lowest in the parallel plating group (0.54 ± 0.19%), followed by the plate-nail (2.89 ± 0.83%) and orthogonal plating groups (3.04 ± 0.51%). Conclusion: Orthogonal plating demonstrated comparable baseline stiffness to plate-nail and parallel plating constructs, and similar biomechanical performance in fatigue loading to plate-nail constructs. All specimens had ≤3% strain after fatigue loading, suggesting sufficient stability for fracture healing. The benefits of enhanced stability from dual-implant fixation may be achieved through orthogonal plating while avoiding an additional medial surgical approach, and therefore warrants further investigation as a novel alternative for distal femur fracture fixation.

Galectin-3 and RAGE differentially control advanced glycation endproduct-induced collagen damage in murine intervertebral disc organ culture.

Background: Back and neck pain are leading causes of global disability that are associated with intervertebral disc (IVD) degeneration. Causes of IVD degeneration are multifactorial, and diet, age, and diabetes have all been linked to IVD degeneration. Advanced glycation endproducts (AGEs) accumulate in the IVD as a result of aging, diet, and diabetes, and AGE accumulation in the IVD has been shown to induce oxidative stress and catabolic activity that result in collagen damage. An association between AGE accumulation and IVD degeneration is emerging, yet mechanism behind this association remains unclear. The Receptor for AGEs (RAGE) is thought to induce catabolic responses in the IVD, and the AGE receptor Galectin 3 (Gal3) had a protective effect in other tissue systems but has not been evaluated in the IVD. Methods: This study used an IVD organ culture model with genetically modified mice to analyze the roles of RAGE and Gal3 in an AGE challenge. Results: Gal3 was protective against an AGE challenge in the murine IVD ex vivo, limiting collagen damage and biomechanical property changes. Gal3 receptor levels in the AF significantly decreased upon an AGE challenge. RAGE was necessary for AGE-induced collagen damage in the IVD, and RAGE receptor levels in the AF significantly increased upon AGE challenge. Discussion: These findings suggest both RAGE and Gal3 are important in the IVD response to AGEs and highlight Gal3 as an important receptor with protective effects on collagen damage. This research improves understanding the mechanisms of AGE-induced IVD degeneration and suggests Gal3 receptor modulation as a potential target for preventative and therapeutic treatment for IVD degeneration.

Lumbar endplate microfracture injury induces Modic-like changes, intervertebral disc degeneration and spinal cord sensitization - An In Vivo Rat Model.

BACKGROUND CONTEXT : Endplate (EP) injury plays critical roles in painful IVD degeneration since Modic changes (MCs) are highly associated with pain. Models of EP microfracture that progress to painful conditions are needed to better understand pathophysiological mechanisms and screen therapeutics. PURPOSE : Establish in vivo rat lumbar EP microfracture model with painful phenotype. STUDY DESIGN/SETTING : In vivo rat study to characterize EP-injury model with characterization of IVD degeneration, vertebral bone marrow remodeling, spinal cord sensitization, and pain-related behaviors. METHODS : EP-driven degeneration was induced in 5-month-old male Sprague-Dawley rats L4-5 and L5-6 IVDs through the proximal vertebral body injury with intradiscal injections of TNFα (n=7) or PBS (n=6), compared to Sham (surgery without EP-injury, n=6). The EP-driven model was assessed for IVD height, histological degeneration, pain-like behaviors (hindpaw von Frey and forepaw grip test), lumbar spine MRI and µCT analyses, and spinal cord substance P (SubP). RESULTS : EP injuries induced IVD degeneration with decreased IVD height and MRI T2 values. EP injury with PBS and TNFα both showed MC type1-like changes on T1 and T2-weighted MRI, trabecular bone remodeling on µCT, and damage in cartilage EP adjacent to the injury. EP injuries caused significantly decreased paw withdrawal threshold and reduced grip forces, suggesting increased pain sensitivity and axial spinal discomfort. Spinal cord dorsal horn SubP was significantly increased, indicating spinal cord sensitization. CONCLUSIONS : EP microfracture can induce crosstalk between vertebral bone marrow, IVD and spinal cord with chronic pain-like conditions. CLINICAL SIGNIFICANCE : This rat EP microfracture model of IVD degeneration was validated to induce MC-like changes and pain-like behaviors that we hope will be useful to screen therapies and improve treatment for EP-drive pain.

Lumbar endplate microfracture injury induces Modic-like changes, intervertebral disc degeneration and spinal cord sensitization - an in vivo rat model.

BACKGROUND CONTEXT: Endplate (EP) injury plays critical roles in painful IVD degeneration since Modic changes (MCs) are highly associated with pain. Models of EP microfracture that progress to painful conditions are needed to better understand pathophysiological mechanisms and screen therapeutics. PURPOSE: Establish in vivo rat lumbar EP microfracture model and assess crosstalk between IVD, vertebra and spinal cord. STUDY DESIGN/SETTING: In vivo rat EP microfracture injury model with characterization of IVD degeneration, vertebral remodeling, spinal cord substance P (SubP), and pain-related behaviors. METHODS: EP-injury was induced in 5 month-old male Sprague-Dawley rats L4-5 and L5-6 IVDs by puncturing through the cephalad vertebral body and EP into the NP of the IVDs followed by intradiscal injections of TNFα (n=7) or PBS (n=6), compared with Sham (surgery without EP-injury, n=6). The EP-injury model was assessed for IVD height, histological degeneration, pain-like behaviors (hindpaw von Frey and forepaw grip test), lumbar spine MRI and µCT, and spinal cord SubP. RESULTS: Surgically-induced EP microfracture with PBS and TNFα injection induced IVD degeneration with decreased IVD height and MRI T2 signal, vertebral remodeling, and secondary damage to cartilage EP adjacent to the injury. Both EP injury groups showed MC-like changes around defects with hypointensity on T1-weighted and hyperintensity on T2-weighted MRI, suggestive of MC type 1. EP injuries caused significantly decreased paw withdrawal threshold, reduced axial grip, and increased spinal cord SubP, suggesting axial spinal discomfort and mechanical hypersensitivity and with spinal cord sensitization. CONCLUSIONS: Surgically-induced EP microfracture can cause crosstalk between IVD, vertebra, and spinal cord with chronic pain-like conditions. CLINICAL SIGNIFICANCE: This rat EP microfracture model was validated to induce broad spinal degenerative changes that may be useful to improve understanding of MC-like changes and for therapeutic screening.

Physiological loading of joints prevents cartilage degradation through CITED2.

Both overuse and disuse of joints up-regulate matrix metalloproteinases (MMPs) in articular cartilage and cause tissue degradation; however, moderate (physiological) loading maintains cartilage integrity. Here, we test whether CBP/p300-interacting transactivator with ED-rich tail 2 (CITED2), a mechanosensitive transcriptional coregulator, mediates this chondroprotective effect of moderate mechanical loading. In vivo, hind-limb immobilization of Sprague-Dawley rats up-regulates MMP-1 and causes rapid, histologically detectable articular cartilage degradation. One hour of daily passive joint motion prevents these changes and up-regulates articular cartilage CITED2. In vitro, moderate (2.5 MPa, 1 Hz) intermittent hydrostatic pressure (IHP) treatment suppresses basal MMP-1 expression and up-regulates CITED2 in human chondrocytes, whereas high IHP (10 MPa) down-regulates CITED2 and increases MMP-1. Competitive binding and transcription assays demonstrate that CITED2 suppresses MMP-1 expression by competing with MMP transactivator, Ets-1 for its coactivator p300. Furthermore, CITED2 up-regulation in vitro requires the p38δ isoform, which is specifically phosphorylated by moderate IHP. Together, these studies identify a novel regulatory pathway involving CITED2 and p38δ, which may be critical for the maintenance of articular cartilage integrity under normal physical activity levels.

Ex vivo biomechanical evaluation of Acute lumbar endplate injury and comparison to annulus fibrosus injury in a rat model.

Back pain is often associated with intervertebral disc (IVD) degeneration, and IVD degeneration phenotypes are commonly characterized by annulus fibrosus (AF)-driven and endplate (EP)-driven phenotypes. Few studies of EP injury exist in animal models, even though clinical studies show EP lesions are strongly associated with IVD pathology and pain. This project established an ex-vivo rat lumbar EP injury model and characterized effects of EP injury on motion segment biomechanical properties, as compared to AF injury, a common way of inducing IVD degeneration. Lumbar motion segments (39 total vertebra-IVD-vertebra sections) assigned to Intact (L1/L2), AF injury and EP injury (L3/L4 and L5/L6 randomly selected), and biomechanically tested in axial tension-compression, stress-relaxation and torsional testing in pre-injury and post-injury conditions using a repeated-measures design. EP injury involved superior vertebra endplate puncture transcorporeally and obliquely. AF injury involved mid-line punctures anterior and bilaterally. Axial ROM, tensile stiffness, hysteresis, and neutral zone stiffness were significantly affected by EP injury but not AF injury. Torque range, torsional stiffness and torsional neutral zone stiffness were significantly affected by AF injury but not EP injury. Stress-relaxation fast time constant was decreased for EP injury. EP and AF injuries induced distinct biomechanical changes in lumbar motion segments with EP injury having the largest impact on axial biomechanical properties and AF injury most prominently affecting torsional properties. This study deepens the understanding of biomechanical mechanism of EP-driven low back pain and provides methods and biomechanical characterization for future in vivo studies.

High fat diet causes inferior vertebral structure and function without disc degeneration in RAGE-KO mice.

Back pain and spinal pathologies are associated with obesity in juveniles and adults, yet studies identifying causal relationships are lacking and none investigate sex differences. This study determined if high fat (HF) diet causes structural and functional changes to vertebrae and intervertebral discs (IVDs); if these changes are modulated in mice with systematic ablation for the receptor for advanced glycation endproducts (RAGE-KO); and if these changes are sex-dependent. Wild-type (WT) and RAGE-KO mice were fed a low fat (LF) or HF diet for 12 weeks starting at 6 weeks, representing the juvenile population. HF diet led to weight/fat gain, glucose intolerance, and increased cytokine levels (IL-5, MIG, and RANTES); with less fat gain in RAGE-KO females. Most importantly, HF diet reduced vertebral trabecular bone volume fraction and compressive and shear moduli, without a modifying effect of RAGE-KO, but with a more pronounced effect in females. HF diet caused reduced cortical area fraction only in WT males. Neither HF diet nor RAGE-KO affected IVD degeneration grade. Biomechanical properties of coccygeal motion segments were affected by RAGE-KO but not diet, with some interactions identified. In conclusion, HF diet resulted in inferior vertebral structure and function with some sex differences, no IVD degeneration, and few modifying effects of RAGE-KO. These structural and functional deficiencies with HF diet provide further evidence that diet can affect spinal structures and may increase the risk for spinal injury and degeneration with aging and additional stressors. Back pain and spinal pathologies are associated with obesity in juveniles and adults, yet studies identifying causal relationships are lacking and none investigate sex differences.

Tenomodulin and Chondromodulin-1 Are Both Required to Maintain Biomechanical Function and Prevent Intervertebral Disc Degeneration.

OBJECTIVE: The underlying mechanisms and molecular factors influencing intervertebral disc (IVD) homeostasis and degeneration remain clinically relevant. Tenomodulin (Tnmd) and chondromodulin (Chm1) are antiangiogenic transmembrane glycoproteins, with cleavable C-terminus, expressed by IVD cells that are implicated in the onset of degenerative processes. We evaluate the organ-level biomechanical impact of knocking out Tnmd alone, and Tnmd and Chm1, simultaneously. DESIGN: Caudal (c5-8) and lumbar vertebrae (L1-4) of skeletally mature male and female 9-month-old wildtype (WT), Tnmd knockout (Tnmd-/-), and Tnmd/Chm1 double knockout (Tnmd-/-/Chm-/-) mice were used (n = 9-13 per group). Disc height index (DHI), histomorphological changes, and axial, torsional, creep, and failure biomechanical properties were evaluated. Differences were assessed by one-way ANOVA with post hoc Bonferroni-corrected comparisons (P < 0.05). RESULTS: Tnmd-/-/Chm1-/- IVDs displayed increased DHI and histomorphological scores that indicated increased IVD degeneration compared to the WT and Tnmd-/- groups. Double knockout IVDs required significantly less torque and energy to initiate torsional failure. Creep parameters were comparable between all groups, except for the slow time constant, which indicated faster outward fluid flow. Tnmd-/- IVDs lost fluid faster than the WT group, and this effect was amplified in the double knockout IVDs. CONCLUSION: Knocking out Tnmd and Chm1 affects IVD fluid flow and organ-level biomechanical function and therefore may play a role in contributing to IVD degeneration. Larger effects of the Tnmd and Chm1 double knockout mice compared to the Tnmd single mutant suggest that Chm1 may play a compensatory role in the Tnmd single mutant IVDs.

The effects of core suture purchase on the biomechanical characteristics of a multistrand locking flexor tendon repair: a cadaveric study.

PURPOSE: To determine the effects of suture purchase on work of flexion (WOF), 2-mm gap force, and load to failure on the combination cross-locked cruciate-interlocking horizontal mattress (CLC-IHM) flexor tendon repair in zone II. METHODS: A total of 33 fresh-frozen cadaveric fingers were mounted in a custom jig, and the flexor digitorum profundus of each finger was fixed to the mobile arm of a tensile strength machine. Initial measurements of WOF were obtained. Each tendon was repaired with the CLC core suture, randomly assigned to placement of 3, 5, 7 or 10 mm from the cut edge of the tendon, and completed with the IHM circumferential suture. After the repair was completed, measurements of WOF were repeated. Each finger was cycled 1000 times. After each 250 cycles, gapping was recorded, and WOF was measured again. Change in WOF (WOF after repair - WOF of intact tendon) was calculated. Tendons were then dissected from the fingers and linearly tested for 2-mm gap force and ultimate load to failure. RESULTS: The group repaired at 10 mm had the lowest percent increase in WOF (5.2%), the highest 2-mm gap force (89.8 N), and the highest ultimate load to failure (111.5 N). The group repaired at 3 mm had the highest percent increase in WOF (22.1%), the lowest 2-mm gap force (54.6 N), and the lowest ultimate load to failure (84.6 N). CONCLUSIONS: A 10-mm suture purchase is the recommended distance for optimal performance for the CLC-IHM combination repair method. This method with a 10-mm suture purchase has a low increase in WOF, high strength, and high resistance to gapping, and it should be strong enough to tolerate early motion.

Males and females exhibit distinct relationships between intervertebral disc degeneration and pain in a rat model.

Back pain is linked to intervertebral disc (IVD) degeneration, but clinical studies show the relationship is complex. This study assessed whether males and females have distinct relationships between IVD degeneration and pain using an in vivo rat model. Forty-eight male and female Sprague-Dawley rats had lumbar IVD puncture or sham surgery. Six weeks after surgery, IVDs were evaluated by radiologic IVD height, histological grading, and biomechanical testing. Pain was assessed by von Frey assay and dorsal root ganglia (DRG) expression of Calca and Tac1 genes. Network analysis visualized which measures of IVD degeneration most related to pain by sex. In both females and males, annular puncture induced structural IVD degeneration, but functional biomechanical properties were similar to sham. Females and males had distinct differences in mechanical allodynia and DRG gene expression, even though sex differences in IVD measurements were limited. Network analysis also differed by sex, with more associations between annular puncture injury and pain in the male network. Sex differences exist in the interactions between IVD degeneration and pain. Limited correlation between measures of pain and IVD degeneration highlights the need to evaluate pain or nociception in IVD degeneration models to better understand nervous system involvement in discogenic pain.

Ex-vivo biomechanics of repaired rat intervertebral discs using genipin crosslinked fibrin adhesive hydrogel.

Microdiscectomy is the current standard surgical treatment for intervertebral disc (IVD) herniation, however annulus fibrosus (AF) defects remain unrepaired which can alter IVD biomechanical properties and lead to reherniation, IVD degeneration and recurrent back pain. Genipin-crosslinked fibrin (FibGen) hydrogel is an injectable AF sealant previously shown to partially restore IVD motion segment biomechanical properties. A small animal model of herniation and repair is needed to evaluate repair potential for early-stage screening of IVD repair strategies prior to more costly large animal and eventual human studies. This study developed an ex-vivo rat caudal IVD herniation model and characterized torsional, axial tension-compression and stress relaxation biomechanical properties before and after herniation injury with or without repair using FibGen. Injury group involved an annular defect followed by removal of nucleus pulposus tissue to simulate a severe herniation while Repaired group involved FibGen injection. Injury significantly altered axial range of motion, neutral zone, torsional stiffness, torque range and stress-relaxation biomechanical parameters compared to Intact. FibGen repair restored the stress-relaxation parameters including effective hydraulic permeability indicating it effectively sealed the IVD defect, and there was a trend for improved tensile stiffness and axial neutral zone length. This study demonstrated a model for studying IVD herniation injury and repair strategies using rat caudal IVDs ex-vivo and demonstrated FibGen sealed IVDs to restore water retention and IVD pressurization. This ex-vivo small animal model may be modified for future in-vivo studies to screen IVD repair strategies using FibGen and other IVD repair biomaterials as an augment to additional large animal and human IVD testing.

Measuring the neutral zone of spinal motion segments: Comparison of multiple analysis methods to quantify spinal instability.

PURPOSE: Neutral zone (NZ) parameters in spinal biomechanics studies are sensitive to spinal instability, disc degeneration, and repair. Multiple methods in the literature quantify NZ, yet no consensus exists on applicability and comparability of methods. This study compares five different NZ quantification methods using two different load-deflection profiles. METHODS: Rat caudal and lumbar motion segments were tested in axial rotation to generate load-deflection curves with profiles exhibiting prominent distinction between elastic and NZ regions (ie, triphasic) and profiles that did not (ie, viscoelastic). NZ was quantified using five methods: trilinear, double sigmoid (DS), zero load, stiffness threshold (ST), and extrapolated elastic zone. Absolute agreement and consistency of NZ parameters were assessed using intraclass correlation (ICC), Bland-Altman analyses, and analysis of variance. RESULTS: For triphasic profiles, NZ magnitude exhibited high consistency (methods correlate but differ in absolute values), and only some methods exhibited agreement. For viscoelastic profiles, NZ magnitude showed limited consistency and no absolute agreement. NZ stiffness had high agreement and consistency across most methods and profiles. For triphasic profiles, the linear NZ regions for all methods were not well-described by a linear fit yet for viscoelastic profiles all methods characterized a linear NZ region. CONCLUSION: This NZ comparison study showed surprisingly limited agreement and consistency among NZ parameters with approximately 5% to 100% difference depending on the method and load-deflection profile. Nevertheless, the DS and ST methods appeared to be most comparable. We conclude that most NZ quantification methods cannot be applied interchangeably, highlighting a need to clearly state NZ calculation methods. Future studies are required to identify which methods are most sensitive to disc degeneration and repair in order to identify a "best" method.

Development of a two-part biomaterial adhesive strategy for annulus fibrosus repair and ex vivo evaluation of implant herniation risk.

Intervertebral disc (IVD) herniation causes pain and disability, but current discectomy procedures alleviate pain without repairing annulus fibrosus (AF) defects. Tissue engineering strategies seal AF defects by utilizing hydrogel systems to prevent recurrent herniation, however current biomaterials are limited by poor adhesion to wetted tissue surfaces or low failure strength resulting in considerable risk of implant herniation upon spinal loading. Here, we developed a two-part repair strategy comprising a dual-modified (oxidized and methacrylated) glycosaminoglycan that can chemically adsorb an injectable interpenetrating network hydrogel composed of fibronectin-conjugated fibrin and poly (ethylene glycol) diacrylate (PEGDA) to covalently bond the hydrogel to AF tissue. We show that dual-modified hyaluronic acid imparts greater adhesion to AF tissue than dual-modified chondroitin sulfate, where the degree of oxidation is more strongly correlated with adhesion strength than methacrylation. We apply this strategy to an ex vivo bovine model of discectomy and demonstrate that PEGDA molecular weight tunes hydrogel mechanical properties and affects herniation risk, where IVDs repaired with low-modulus hydrogels composed of 20kDa PEGDA failed at levels at or exceeding discectomy, the clinical standard of care. This strategy bonds injectable hydrogels to IVD extracellular matrix proteins, is optimized to seal AF defects, and shows promise for IVD repair.

Fibrin-Genipin Hydrogel for Cartilage Tissue Engineering in Nasal Reconstruction.

OBJECTIVES: Nasal reconstruction is limited by the availability of autologous cartilage. The aim is to investigate an adhesive biomaterial for tissue engineering of nasal cartilage by evaluating mechanical properties of hydrogels made of fibrin crosslinked with genipin as compared to native tissue. METHODS: Hydrogels of fibrin, fibrin-genipin, and fibrin-genipin with extracellular matrix (ECM) particles were created and evaluated with mechanical testing to determine compression, tensile, and shear properties. Rabbit nasal septal cartilage was harvested and tested in these modalities for comparison. Transmission electron microscopy characterized hydrogel structure. RESULTS: Fibrin-genipin gels had higher compressive, tensile, and shear moduli compared to fibrin alone or fibrin-genipin with ECM. However, all hydrogel formulations had lower moduli than the rabbit nasal septal cartilage. Electron microscopy showed genipin crosslinking increased structural density of the hydrogel and that cartilage ECM created larger structural features with lower crosslinking density. CONCLUSION: The addition of genipin significantly improved mechanical properties of fibrin hydrogels by increasing the compressive, tensile, and shear moduli. The addition of cartilage ECM, which can add native structure and composition, resulted in decreased moduli values. Fibrin-genipin is a bioactive and biomechanically stable hydrogel that may offer promise as a scaffold for cartilage tissue engineering in nasal reconstruction, yet further augmentation is required to match material properties of native nasal cartilage.