Neo-Natal Castration Leads to Subtle Differences in Porcine Anterior Cruciate Ligament Morphology and Function in Adolescence.

Female adolescent athletes are at a higher risk of tearing their anterior cruciate ligament (ACL) than male counterparts. While most work related to hormones has focused on the effects of estrogen to understand the increased risk of ACL injury, there are other understudied factors, including testosterone. The purpose of this study was to determine how surgical castration in the male porcine model influences ACL size and function across skeletal growth. Thirty-six male Yorkshire crossbreed pigs were raised to 3 (juvenile), 4.5 (early adolescent), and 6 months (adolescent) of age. Animals were either castrated (barrows) within 2 weeks after birth or were left intact (boars). Posteuthanasia, joint and ACL size were assessed via MRI, and biomechanics were assessed via a robotic testing system. Joint size increased throughout age, yet barrows had smaller joints than boars. ACL cross-sectional area (CSA), length, volume, and in situ stiffness increased with age, as did the percent contribution of the ACL anteromedial (AM) bundle to resisting loads. Boar ACL, AM bundle, and PL bundle volumes were 19%, 25%, and 15% larger than barrows across ages. However, ACL CSA, in situ stiffness, and bundle contribution were similar between boars and barrows. The barrows had smaller temporal increases in AM bundle function than boars, but these data were highly variable. Early and sustained loss in testosterone leads to subtle differences in ACL morphology but may not influence measures associated with increased injury risk, such as CSA or bundle forces in response to applied loads.

Controlled Stiffness of Direct-Write, Near-Field Electrospun Gelatin Fibers Generates Differences in Tenocyte Morphology and Gene Expression.

Tendinopathy is a leading cause of mobility issues. Currently, the cell-matrix interactions involved in the development of tendinopathy are not fully understood. In vitro tendon models provide a unique tool for addressing this knowledge gap as they permit fine control over biochemical, micromechanical, and structural aspects of the local environment to explore cell-matrix interactions. In this study, direct-write, near-field electrospinning of gelatin solution was implemented to fabricate micron-scale fibrous scaffolds that mimic native collagen fiber size and orientation. The stiffness of these fibrous scaffolds was found to be controllable between 1 MPa and 8 MPa using different crosslinking methods (EDC, DHT, DHT+EDC) or through altering the duration of crosslinking with EDC (1 h to 24 h). EDC crosslinking provided the greatest fiber stability, surviving up to 3 weeks in vitro. Differences in stiffness resulted in phenotypic changes for equine tenocytes with low stiffness fibers (∼1 MPa) promoting an elongated nuclear aspect ratio while those on high stiffness fibers (∼8 MPa) were rounded. High stiffness fibers resulted in the upregulation of matrix metalloproteinase (MMPs) and proteoglycans (possible indicators for tendinopathy) relative to low stiffness fibers. These results demonstrate the feasibility of direct-written gelatin scaffolds as tendon in vitro models and provide evidence that matrix mechanical properties may be crucial factors in cell-matrix interactions during tendinopathy formation.

A Systems Approach to Biomechanics, Mechanobiology, and Biotransport.

The human body represents a collection of interacting systems that range in scale from nanometers to meters. Investigations from a systems perspective focus on how the parts work together to enact changes across spatial scales, and further our understanding of how systems function and fail. Here, we highlight systems approaches presented at the 2022 Summer Biomechanics, Bio-engineering, and Biotransport Conference in the areas of solid mechanics; fluid mechanics; tissue and cellular engineering; biotransport; and design, dynamics, and rehabilitation; and biomechanics education. Systems approaches are yielding new insights into human biology by leveraging state-of-the-art tools, which could ultimately lead to more informed design of therapies and medical devices for preventing and treating disease as well as rehabilitating patients using strategies that are uniquely optimized for each patient. Educational approaches can also be designed to foster a foundation of systems-level thinking.

A Direct Comparison of Node and Element-Based Finite Element Modeling Approaches to Study Tissue Growth.

Finite element analysis is a useful tool to model growth of biological tissues and predict how growth can be impacted by stimuli. Previous work has simulated growth using node-based or element-based approaches, and this implementation choice may influence predicted growth, irrespective of the applied growth model. This study directly compared node-based and element-based approaches to understand the isolated impact of implementation method on growth predictions by simulating growth of a bone rudiment geometry, and determined what conditions produce similar results between the approaches. We used a previously reported node-based approach implemented via thermal expansion and an element-based approach implemented via osmotic swelling, and we derived a mathematical relationship to relate the growth resulting from these approaches. We found that material properties (modulus) affected growth in the element-based approach, with growth completely restricted for high modulus values relative to the growth stimulus, and no restriction for low modulus values. The node-based approach was unaffected by modulus. Node- and element-based approaches matched marginally better when the conversion coefficient to relate the approaches was optimized based on the results of initial simulations, rather than using the theoretically predicted conversion coefficient (median difference in node position 0.042 cm versus 0.052 cm, respectively). In summary, we illustrate here the importance of the choice of implementation approach for modeling growth, provide a framework for converting models between implementation approaches, and highlight important considerations for comparing results in prior work and developing new models of tissue growth.

Biomechanical evaluation of a novel barbed suture pattern with epitendinous suture augmentation in a canine flexor tendon model.

OBJECTIVE: To determine the effect of a novel barbed suture pattern (NBSP) compared to a three-loop-pulley (3LP) with and without epitendinous suture (ES) augmentation on the biomechanical strength and gap formation of repaired canine tendons. STUDY DESIGN: Ex vivo, cadaveric, randomized, experimental study. SAMPLE POPULATION: Forty, adult superficial digital flexor tendons (SDFT). METHODS: SDFT were randomly assigned to one of four groups (n = 10/group). Sharp tenotomy was performed and repaired with 3LP, NBSP, 3LP + ES, and NBSP + ES. Constructs were tested to failure while evaluating yield, peak, and failure loads, loads at 1 and 3 mm gap formation, and failure mode. RESULTS: Constructs augmented with ES sustained 80% greater yield (p < .001), peak (p < .001), and failure (p < .001) loads, with no difference between 3LP + ES and NBSP + ES constructs regarding peak (p = .614), and failure forces (p = .865). Loads resulting in 1 and 3 mm gap formation were greater when constructs were augmented with an ES (p ≤ .003). Failure mode differed between groups (p < .001), occurring predominantly due to suture pull-through in 3LP and NBSP groups compared to tissue failure distant to the repair site in ES augmented constructs. CONCLUSION: Tendons repaired with the NBSP used in this study resisted similar forces as those repaired with 3LP. Augmentation with an ES improved the biomechanical properties of repaired constructs, including resistance to gap formation. CLINICAL RELEVANCE: The NBSP repair tested here may be advantageous over monofilament suture repair as it uses a similar-sized barbed core suture but eliminates the requirement for knot tying.

Biomechanical analysis of accessory tendon graft augmentation for primary gastrocnemius tendon reconstruction in dogs.

OBJECTIVE: To evaluate the effect of accessory tendon graft (ATG) augmentation as an adjunct to a core locking-loop (LL) and epitendinous suture (ES) repair in a gastrocnemius tendon (GT) model. STUDY DESIGN: Randomized, ex vivo, biomechanical. POPULATION: Twenty-two canine GT musculotendinous constructs. METHODS: GT repair constructs were randomly divided into two groups (n = 10/group). After transection, paired GT were repaired with LL + ES alone or with concurrent ATG augmentation. Yield, peak and failure loads, tensile loads required to create 1 and 3 mm gapping, and failure modes were evaluated. Four GT were used as intact controls for validation of testing methodology. ATG constructs were compared to LL + ES and control specimens. RESULTS: Yield (p < .0001), peak (p = .0001) and failure loads (p = .0003) were greater when ATG was used for repair. Greater force was required to cause 1 mm (p = .0001) and 3 mm (p = .0002) gap formation in the ATG group, however, the frequency of gap formation did not differ between groups. All repaired constructs failed exclusively by suture pull-through. CONCLUSION: Autologous ATG augmentation as an adjunct to primary GT repair increased yield, peak and failure forces by approximately 1.6×, 1.9×, 1.8× respectively and required 2.1× greater force to cause 1 and 3 mm formation respectively compared to LL + ES repairs alone. CLINICAL SIGNIFICANCE: ATG augmentation should be considered as an autologous method to support and strengthen the primary GT repair. These results justify studies to determine the effect of ATG on clinical function following graft harvest in dogs.

Ex vivo evaluation of novel core tenorrhaphy patterns in dogs.

OBJECTIVE: To compare the biomechanical properties and gapping characteristics of four novel tenorrhaphy patterns in a canine flexor tendon model. STUDY DESIGN: Ex vivo, randomized, biomechanical study. SAMPLE POPULATION: Superficial digital flexor tendons of 60 forelimbs (30 dogs). METHODS: Each tendon was transected 25 mm distal to its musculotendinous junction prior to tenorrhaphy with 2-0 polypropylene. Repair patterns included the three-loop pulley (3LP, control), exposed double-cross-lock (ExDCrL), embedded double-cross-lock (EmDCrL), triple-circle-lock (TCiL), and Modified-Tang patterns (MTang) were randomly assigned to each experimental group (n = 12/group). Yield, peak, and failure loads, gap formation and failure modes were compared. RESULTS: Tendons repaired with ExDCrL (p < .0001), EmDCrL (p < .0001), and MTang (p < .0001) sustained yield, peak, and failure loads ~2.2x, ~2.0x, and ~1.9x, respectively, greater than those repaired with 3LP. Loads to 1 and 3 mm gapping were also higher for ExDCrL (p < .0001), EmDCrL (p < .0004), and MTang constructs (p < .0017) compared to 3LP. Although TCiL constructs sustained higher loads, their resistance to gap formation did not differ from that of 3LP repairs. Failure mode differed between groups (p < .0001), EmDCrL, ExDCrL, MTang, and TCiL constructs failing predominantly by suture breakage compared to 3LP repairs that failed by suture pull-through. CONCLUSION: Use of novel patterns ExDCrL, EmDCrL, and MTang improved resistance to loads and gap formation and were biomechanically superior compared to 3LP in healthy canine tendon repairs. CLINICAL SIGNIFICANCE: These results justify in vivo evaluation of ExDCrL, EmDCrL, or MTang pattern for tenorrhaphy in dogs.

Mechanical properties of tissue formed in vivo are affected by 3D-bioplotted scaffold microarchitecture and correlate with ECM collagen fiber alignment.

Purpose: Musculoskeletal soft tissues possess highly aligned extracellular collagenous networks that provide structure and strength. Such an organization dictates tissue-specific mechanical properties but can be difficult to replicate by engineered biological substitutes. Nanofibrous electrospun scaffolds have demonstrated the ability to control cell-secreted collagen alignment, but concerns exist regarding their scalability for larger and anatomically relevant applications. Additive manufacturing processes, such as melt extrusion-based 3D-Bioplotting, allow fabrication of structurally relevant scaffolds featuring highly controllable porous microarchitectures.Materials and Methods: In this study, we investigate the effects of 3D-bioplotted scaffold design on the compressive elastic modulus of neotissue formed in vivo in a subcutaneous rat model and its correlation with the alignment of ECM collagen fibers. Polycaprolactone scaffolds featuring either 100 or 400 µm interstrand spacing were implanted for 4 or 12 weeks, harvested, cryosectioned, and characterized using atomic-force-microscopy-based force mapping.Results: The compressive elastic modulus of the neotissue formed within the 100 µm design was significantly higher at 4 weeks (p < 0.05), but no differences were observed at 12 weeks. In general, the tissue stiffness was within the same order of magnitude and range of values measured in native musculoskeletal soft tissues including the porcine meniscus and anterior cruciate ligament. Finally, a significant positive correlation was noted between tissue stiffness and the degree of ECM collagen fiber alignment (p < 0.05) resulting from contact guidance provided by scaffold strands.Conclusion: These findings demonstrate the significant effects of 3D-bioplotted scaffold microarchitectures in the organization and sub-tissue-level mechanical properties of ECM in vivo.

Effect of partial vs complete circumferential epitendinous suture placement on the biomechanical properties and gap formation of canine cadaveric tendons.

OBJECTIVE: To determine the effect of partial vs complete circumferential epitendinous suture (ES) placement in addition to a core suture on the biomechanical strength and gapping characteristics of repaired canine tendinous constructs. STUDY DESIGN: Ex vivo, biomechanical study. SAMPLE POPULATION: Thirty-six canine superficial digital flexor tendons. METHODS: Superficial digital flexor tendons were randomly assigned to three groups (n = 12), sharply transected and repaired with a core locking-loop suture with Group 1 a partial circumferential ES, 180° on the palmar side; Group 2 a complete circumferential ES, 360° and double knotting technique; or Group 3 a complete circumferential ES, 360° and single knotting technique. After preloading, constructs were distracted to monotonic failure. Failure mode, gap formation, yield, peak, and failure forces were analyzed. RESULTS: Mean yield (group 1 = 68.6 N, group 2 = 106.5 N, group 3 = 114 N, P < .013), peak (group 1 = 92.8 N, group 2 = 134.6 N, group 3 = 147.3 N; P < .001), and failure (group 1 = 88.7 N, group 2 = 133.0 N, group 3 = 145.5 N, P < .001) loads differed between groups. No difference in yield (P = .874), peak (P = .434), or failure load (P = .434) was detected between complete circumferential ES groups. Force to create 1-mm (P < .001) and 3-mm (P < .038) gap formation was greater in specimens with complete vs partial circumferential ES placement. Complete circumferential ES repairs failed primarily by suture pull-through compared with suture breakage in most partial circumferential ES constructs. CONCLUSION: Addition of a complete circumferential ES with a single or double knotting technique increased the biomechanical strength of normal tendon repairs while reducing gap formation compared with partial ES placement alone. CLINICAL SIGNIFICANCE: Complete circumferential ES is recommended over partial ES placement.

Biomechanical evaluation of an autologous flexor digitorum lateralis graft to augment the surgical repair of gastrocnemius tendon laceration in a canine ex vivo model.

OBJECTIVE: To evaluate the effect of an autologous flexor digitorum lateralis (FDL) graft to augment a three-loop pulley (3LP) core repair in a canine cadaveric gastrocnemius tendon (GT) laceration model. STUDY DESIGN: Ex vivo, biomechanical study. SAMPLE POPULATION: Twenty-six canine cadaveric hind limbs. METHODS: Tendons were divided into two groups (n = 13). After sharp transection, paired GT were repaired with 3LP or 3LP + FDL tendon augmentation. Yield, peak and failure loads, tensile loads required to create 1 and 3-mm gapping, and failure modes were analyzed. Significance was set at P < .05. RESULTS: Yield and failure force (mean ± SD) for 3LP + FDL were 134.9 ± 44.1 N and 205.4 ± 46.4 N, respectively, which were greater than for 3LP alone (67.9 ± 12.2 N and 91.8 ± 9.9 N, respectively, P < .0001). No constructs (0%) formed 1 or 3-mm gaps in the 3LP + FDL graft group compared with 84% and 39% for 3LP, respectively (P < .0001). Failure modes were different between groups (P < .001), with 85% of 3LP + FDL constructs failing by tissue rupture at the myotendinous junction, distant to the repair site. CONCLUSION: Addition of an autologous FDL graft to a core 3LP tendon repair increased yield, peak, and failure forces by twofold, 2.3-fold, and 2.2-fold, respectively, compared with core 3LP alone while preventing the occurrence of gap formation. CLINICAL SIGNIFICANCE: Use of FDL tendon augmentation for GT laceration may increase repair site strength and resist gap formation better than 3LP core suture use alone. Additional studies are required in vivo to determine the effect of FDL graft augmentation on clinical function.

Influence of barbed epitendinous sutures combined with a core locking-loop suture to repair experimental flexor tendon lacerations.

OBJECTIVE: To determine the influence of barbed epitendinous sutures (ES) on the biomechanical properties and gap formation of repaired canine tendons. STUDY DESIGN: Ex vivo, experimental study. SAMPLE POPULATION: Eighty (n = 16/group) canine superficial digital flexor tendons (SDFT). METHODS: After transection, SDFT were repaired with a locking-loop (LL) pattern alone (group 1), an LL + smooth ES with monofilament suture (group 2), an LL + V-loc-ES (group 3), an LL + Quill-ES (group 4), or an LL + Stratafix-ES (group 5). All core LL repairs were performed with 0 USP polypropylene, and all ES were placed with 2-0 USP equivalent. Constructs were preloaded and tested to failure. Yield, peak, and failure loads; occurrence of gap formation; and failure modes were compared. RESULTS: Yield loads were greater for groups 2 and 5 (P < .0001). Peak and failure loads were greater when an ES was used (P < .005), especially for groups 2 and 5 (P < .0001). Groups with an ES required higher loads to generate 1- and 3-mm gaps compared with specimens without an ES (P < .002). Force to create 1- and 3-mm gaps was greater for group 5 (P < .0001) and groups 2 and 5 (P < .0001), respectively. Failure mechanism did not differ (P = .092) between ES groups, consisting of suture breakage in 51 of 64 constructs compared with pull-through in seven of 16 group 1 constructs. CONCLUSION: Epitendinous suture placement improved the biomechanical properties of repaired tendons. Stratafix barbed suture performed better as an ES compared with other barbed sutures and similarly to monofilament suture. CLINICAL SIGNIFICANCE: Stratafix barbed suture eliminates the requirement for knot tying and seems to be equivalent to smooth monofilament suture when used as an ES in this pattern.

In Situ Joint Stiffness Increases During Skeletal Growth but Decreases Following Partial and Complete Anterior Cruciate Ligament Injury.

Partial and complete anterior cruciate ligament (ACL) injuries occur in both pediatric and adult populations and can result in loss of joint stability and function. The sigmoidal shape of knee joint function (load-translation curve) under applied loads includes a low-load region (described by slack length) followed by a high-load region (described by stiffness). However, the impact of age and injury on these parameters is not fully understood. The current objective was to measure the effects of age and injury on the shape of joint function in a porcine model. In response to an applied anterior-posterior tibial load, in situ slack did not change (p > 0.05), despite sevenfold increases in joint size with increasing age. Joint stiffness increased from an average of 10 N/mm in early youth to 47 N/mm in late adolescence (p < 0.05). In situ ACL stiffness increased similarly, and changes in in situ joint stiffness and ACL stiffness were highly correlated across ages. With complete ACL injury, in situ slack length increased by twofold to fourfold and in situ stiffness decreased threefold to fourfold across ages (p < 0.05). Partial ACL injury resulted in less dramatic, but statistically significant, increases in joint slack and significant decreases in in situ joint stiffness in the adolescent age groups (p < 0.05). This work furthers our understanding of the interaction between joint biomechanics and ACL function throughout growth and the impact of ACL injury in the skeletally immature joint.

Biomechanical Function and Size of the Anteromedial and Posterolateral Bundles of the ACL Change Differently with Skeletal Growth in the Pig Model.

BACKGROUND: ACL injuries are becoming increasingly common in children and adolescents, but little is known regarding age-specific ACL function in these patients. To improve our understanding of changes in musculoskeletal tissues during growth and given the limited availability of pediatric human cadaveric specimens, tissue structure and function can be assessed in large animal models, such as the pig. QUESTIONS/PURPOSES: Using cadaveric porcine specimens ranging throughout skeletal growth, we aimed to assess age-dependent changes in (1) joint kinematics under applied AP loads and varus-valgus moments, (2) biomechanical function of the ACL under the same loads, (3) the relative biomechanical function of the anteromedial and posterolateral bundles of the ACL; and (4) size and orientation of the anteromedial and posterolateral bundles. METHODS: Stifle joints (analogous to the human knee) were collected from female Yorkshire crossbreed pigs at five ages ranging from early youth to late adolescence (1.5, 3, 4.5, 6, and 18 months; n = 6 pigs per age group, 30 total), and MRIs were performed. A robotic testing system was used to determine joint kinematics (AP tibial translation and varus-valgus rotation) and in situ forces in the ACL and its bundles in response to applied anterior tibial loads and varus-valgus moments. To see if morphological changes to the ACL compared with biomechanical changes, ACL and bundle cross-sectional area, length, and orientation were calculated from MR images. RESULTS: Joint kinematics decreased with increasing age. Normalized AP tibial translation decreased by 44% from 1.5 months (0.34 ± 0.08) to 18 months (0.19 ± 0.02) at 60° of flexion (p < 0.001) and varus-valgus rotation decreased from 25° ± 2° at 1.5 months to 6° ± 2° at 18 months (p < 0.001). The ACL provided the majority of the resistance to anterior tibial loading at all age groups (75% to 111% of the applied anterior force; p = 0.630 between ages). Anteromedial and posterolateral bundle function in response to anterior loading and varus torque were similar in pigs of young ages. During adolescence (4.5 to 18 months), the in situ force carried by the anteromedial bundle increased relative to that carried by the posterolateral bundle, shifting from 59% ± 22% at 4.5 months to 92% ± 12% at 18 months (data for 60° of flexion, p < 0.001 between 4.5 and 18 months). The cross-sectional area of the anteromedial bundle increased by 30 mm throughout growth from 1.5 months (5 ± 2 mm) through 18 months (35 ± 8 mm; p < 0.001 between 1.5 and 18 months), while the cross-sectional area of the posterolateral bundle increased by 12 mm from 1.5 months (7 ± 2 mm) to 4.5 months (19 ± 5 mm; p = 0.004 between 1.5 and 4.5 months), with no further growth (17 ± 7 mm at 18 months; p = 0.999 between 4.5 and 18 months). However, changes in length and orientation were similar between the bundles. CONCLUSION: We showed that the stifle joint (knee equivalent) in the pig has greater translational and rotational laxity in early youth (1.5 to 3 months) compared with adolescence (4.5 to 18 months), that the ACL functions as a primary stabilizer throughout growth, and that the relative biomechanical function and size of the anteromedial and posterolateral bundles change differently with growth. CLINICAL RELEVANCE: Given the large effects observed here, the age- and bundle-specific function, size, and orientation of the ACL may need to be considered regarding surgical timing, graft selection, and graft placement. In addition, the findings of this study will be used to motivate pre-clinical studies on the impact of partial and complete ACL injuries during skeletal growth.

Engineering 3D-Bioplotted scaffolds to induce aligned extracellular matrix deposition for musculoskeletal soft tissue replacement.

PURPOSE: Tissue engineering and regenerative medicine approaches have the potential to overcome the challenges associated with current treatment strategies for meniscus injuries. 3D-Bioplotted scaffolds are promising, but have not demonstrated the ability to guide the formation of aligned collagenous matrix in vivo, which is critical for generating functional meniscus tissue. In this study, we evaluate the ability of 3D-Bioplotted scaffold designs with varying interstrand spacing to induce the deposition of aligned matrix in vivo. MATERIALS AND METHODS: 3D-Bioplotted polycaprolactone scaffolds with 100, 200, or 400 µm interstrand spacing were implanted subcutaneously in a rat model for 4, 8, or 12 weeks. Scaffolds were harvested, paraffin-embedded, sectioned, and stained to visualize cell nuclei and collagen. Quantitative image analysis was used to evaluate cell density, matrix fill, and collagen fiber alignment within the scaffolds. RESULTS: By 4 weeks, cells had infiltrated the innermost scaffold regions. Similarly, collagenous matrix filled interstrand regions nearly completely by 4 weeks. By 12 weeks, aligned collagen was present in all scaffolds. Generally, alignment along the scaffold strands increased over time for all three interstrand spacing groups. Distribution of collagen fiber alignment angles narrowed as interstrand spacing decreased. CONCLUSIONS: 3D-Bioplotted scaffolds allow for complete cell infiltration and collagenous matrix production throughout the scaffold. The ability to use interstrand spacing as a means of controlling the formation of aligned collagen in vivo was demonstrated, which helps establish a design space for scaffold-based meniscus tissue engineering.

Neo-natal castration leads to subtle differences in porcine anterior cruciate ligament morphology and function in adolescence.

Female adolescent athletes are at a higher risk of tearing their anterior cruciate ligament (ACL) than male counterparts. While most work related to hormones has focused on the effects of estrogen to understand the increased risk of ACL injury, there are other understudied factors, including testosterone. The purpose of this study was to determine how surgical castration in the male porcine model influences ACL size and function across skeletal growth. Thirty-six male Yorkshire crossbreed pigs were raised to 3 (juvenile), 4.5 (early adolescent), and 6 months (adolescent) of age. Animals were either castrated (barrows) within 1-2 weeks after birth or were left intact (boars). Post-euthanasia, joint and ACL size were assessed via MRI, and biomechanics were assessed via a robotic testing system. Joint size increased throughout age, yet barrows had smaller joints than boars (p<0.001 for all measures). ACL cross-sectional area (CSA), length, volume, and stiffness increased with age (p<0.0001), as did ACL anteromedial (AM) bundle percent contribution to resisting loads (p=0.012). Boar ACL, AM bundle, and PL bundle volumes were 19% (p=0.002), 25% (p=0.003), and 15% (p=0.04) larger than barrows across ages. However, CSA, stiffness, and bundle contribution were similar between boars and barrows (p>0.05). The barrows had smaller temporal increases in AM bundle percent function than boars, but these data were highly variable. Thus, early and sustained loss in testosterone leads to subtle differences in ACL morphology, but may not influence measures associated with increased injury risk, such as CSA or bundle forces in response to applied loads.

Towards a standardized multi-tissue decellularization protocol for the derivation of extracellular matrix materials.

The goal of tissue decellularization is to efficiently remove unwanted cellular components, such as DNA and cellular debris, while retaining the complex structural and molecular milieu within the extracellular matrix (ECM). Decellularization protocols to date are centered on customized tissue-specific and lab-specific protocols that involve consecutive manual steps which results in variable and protocol-specific ECM material. The differences that result from the inconsistent protocols between decellularized ECMs affect consistency across batches, limit comparisons between results obtained from different laboratories, and could limit the transferability of the material for consistent laboratory or clinical use. The present study is the first proof-of-concept towards the development of a standardized protocol that can be used to derive multiple ECM biomaterials (powders and hydrogels) via a previously established automated system. The automated decellularization method developed by our group was used due to its short decellularization time (4 hours) and its ability to reduce batch-to-batch variability. The ECM obtained using this first iteration of a unified protocol was able to produce ECM hydrogels from skin, lung, muscle, tendons, cartilage, and laryngeal tissues. All hydrogels formed in this study were cytocompatible and showed gelation and rheological properties consistent with previous ECM hydrogels. The ECMs also showed unique proteomic composition. The present study represents the first step towards developing standardized protocols that can be used on multiple tissues in a fast, scalable, and reproducible manner.

Preclinical tendon and ligament models: Beyond the 3Rs (replacement, reduction, and refinement) to 5W1H (why, who, what, where, when, how).

Several tendon and ligament animal models were presented at the 2022 Orthopaedic Research Society Tendon Section Conference held at the University of Pennsylvania, May 5 to 7, 2022. A key objective of the breakout sessions at this meeting was to develop guidelines for the field, including for preclinical tendon and ligament animal models. This review summarizes the perspectives of experts for eight surgical small and large animal models of rotator cuff tear, flexor tendon transection, anterior cruciate ligament tear, and Achilles tendon injury using the framework: "Why, Who, What, Where, When, and How" (5W1H). A notable conclusion is that the perfect tendon model does not exist; there is no single gold standard animal model that represents the totality of tendon and ligament disease. Each model has advantages and disadvantages and should be carefully considered in light of the specific research question. There are also circumstances when an animal model is not the best approach. The wide variety of tendon and ligament pathologies necessitates choices between small and large animal models, different anatomic sites, and a range of factors associated with each model during the planning phase. Attendees agreed on some guiding principles including: providing clear justification for the model selected, providing animal model details at publication, encouraging sharing of protocols and expertise, improving training of research personnel, and considering greater collaboration with veterinarians. A clear path for translating from animal models to clinical practice was also considered as a critical next step for accelerating progress in the tendon and ligament field.

Potential of healing a transected anterior cruciate ligament with genetically modified extracellular matrix bioscaffolds in a goat model.

PURPOSE: Biological augmentation to heal a torn anterior cruciate ligament (ACL) has gained significant interest. This study examined the potential advantages of using extracellular matrix (ECM) bioscaffolds from galactosyl-α(1,3)galactose deficient pigs to heal the transected ACL. METHODS: In 16 skeletally mature goats, the ACL in the right hindlimb was transected and repaired. In 9 of these animals, an ECM sheet was wrapped around the injury site and with an ECM hydrogel injected into the transected site. The remaining 7 animals were treated with suture repair only. The left hindlimb served as a sham-operated control. RESULTS: After 12 weeks, the healing ACL in the ECM-treated group showed an abundance of continuous neo-tissue formation, while only limited tissue growth was found after suture repair only. The cross-sectional area of the ACL from the ECM-treated group was similar to sham-operated controls (n.s.) and was 4.5 times those of the suture repair group (P < 0.05). The stiffness of the femur-ACL-tibia complexes from the ECM-treated group was 2.4 times those of the suture repair group (P < 0.05). Furthermore, these values reached 48% of the sham-operated controls (53 ± 19 N/mm and 112 ± 21 N/mm, respectively, P < 0.05). CONCLUSIONS: The application of an ECM bioscaffold and hydrogel was found to accelerate the healing of a transected ACL following suture repair in the goat model with limited tissue hypertrophy and improvement in some of its biomechanical properties. Although more work is necessary to fully restore the function of the normal ACL, these early results offer a potential new approach to aid ACL healing.

Elucidating the combinatorial effect of substrate stiffness and surface viscoelasticity on cellular phenotype.

Cells maintain tensional homeostasis by monitoring the mechanics of their microenvironment. In order to understand this mechanotransduction phenomenon, hydrogel materials have been developed with either controllable linear elastic or viscoelastic properties. Native biological tissues, and biomaterials used for medical purposes, often have complex mechanical properties. However, due to the difficulty in completely decoupling the elastic and viscous components of hydrogel materials, the effect of complex composite materials on cellular responses has largely gone unreported. Here, we characterize a novel composite hydrogel system capable of decoupling and individually controlling both the bulk stiffness and surface viscoelasticity of the material by combining polyacrylamide (PA) gels with microgel thin films. By taking advantage of the high degree of control over stiffness offered by PA gels and viscoelasticity, in terms of surface loss tangent, of microgel thin films, it is possible to study the influence that bulk substrate stiffness and surface loss tangent have on complex fibroblast responses, including cellular and nuclear morphology and gene expression. This material system provides a facile method for investigating cellular responses to complex material mechanics with great precision and allows for a greater understanding of cellular mechanotransduction mechanisms than previously possible through current model material platforms.

Age- and Sex-Specific Joint Biomechanics in Response to Partial and Complete Anterior Cruciate Ligament Injury in the Porcine Model.

CONTEXT: Pediatric anterior cruciate ligament (ACL) injury rates are increasing and are highest in female adolescents. Complete ACL tears are typically surgically reconstructed, but few guidelines and very limited data exist regarding the need for surgical reconstruction or rehabilitation for partial ACL tears in skeletally immature patients. OBJECTIVE: To evaluate the effects of partial (anteromedial bundle) and complete ACL transection on joint laxity and tissue forces under anterior and rotational loads in male and female stifle joints throughout skeletal growth in the porcine model. DESIGN: Descriptive laboratory study. SETTING: Laboratory. PATIENTS OR OTHER PARTICIPANTS: We studied 60 male and female Yorkshire crossbreed pigs aged 1.5, 3, 4.5, 6, and 18 months (n = 6 pigs per age per sex). MAIN OUTCOME MEASURE(S): Joint laxity was measured in intact, anteromedial bundle-transected, and ACL-transected joints under applied anterior-posterior drawer and varus-valgus torque using a robotic testing system. Loading of the soft tissues in the stifle joint was measured under each condition. RESULTS: Anterior-posterior joint laxity increased by 13% to 50% (P < .05) after anteromedial bundle transection and 75% to 178% (P < .05) after ACL transection. Destabilization after anteromedial bundle transection increased with age (P < .05) and was greater in late female than late male adolescents (P < .05). In anteromedial bundle-transected joints, the posterolateral bundle resisted the anterior load. In ACL-transected joints, the medial collateral ligament (MCL) contribution was largest, followed by the medial meniscus. The MCL contribution was larger and the medial meniscus contribution was smaller in male versus female specimens. CONCLUSIONS: Partial ACL transection resulted in moderate increases in joint laxity, with the remaining bundle performing the primary ACL function. Destabilization due to partial ACL transection (anteromedial bundle) was largest in late adolescent joints, indicating that operative treatment should be considered in active, late-adolescent patients with this injury. Increased forces on the MCL and medial meniscus after ACL transection suggested that rehabilitation protocols may need to focus on protecting these tissues.