Donor Age and Time in Culture Affect Dermal Fibroblast Contraction in an In Vitro Hydrogel Model.

Current cellular hydrogel-based skin grafts composed of human dermal fibroblasts and a hydrogel scaffold tend to minimize contraction of full-thickness skin wounds and support skin regeneration. However, there has been no comparison between the sources of the dermal fibroblast used. Products using human adult or neonatal foreskin dermal fibroblasts are often expanded in vitro and used after multiple passages without a clear understanding of the effects of this initial production step on the quality and reproducibility of the cellular behavior. Based on the known effects of 2D tissue culture expansion on cellular proliferation and gene expression, we hypothesized that differences in donor age and time in culture may influence cellular properties and contractile behavior in a fibroblast-populated collagen matrix. Using porcine skin as a model based on its similarity to human skin in structure and wound healing properties, we isolated porcine dermal fibroblasts of three different donor ages for use in a 2D proliferation assay and in a 3D cell-populated collagen matrix contractility assay. In 2D cell culture, doubling time remained relatively consistent between all age groups from passage 1 to 6. In the contractility assays, fetal and neonatal groups contracted faster and generated more contractile force than the adult group at passage 1 in vitro. However, after five passages in culture, there was no difference in contractility between ages. These results show how cellular responses in a hydrogel scaffold differ based on donor age and time in culture in vitro, and suggest that consistency in the cellular component of bioengineered skin products could be beneficial in the biomanufacturing of consistent, reliable skin grafts and graft in vivo models. Future research and therapies using bioengineered skin grafts should consider how results may vary based on donor age and time in culture before seeding. Impact statement Little is known about the impact of donor cell age and time in culture on the contraction of cellular, hydrogel-based skin grafts. These results show how cellular phenotypes of porcine fibroblasts differ based on donor age and time in culture. This information is beneficial when addressing important inconsistencies in biomanufacturing of bioengineered skin grafts and in vitro models. These findings are relevant to research and therapies using bioengineered skin graft models and the results can be used to increase reproducibility and consistency during the production of bioengineered skin constructs. The information from this study can be extrapolated to future in vivo studies using human dermal fibroblasts in an in vivo model to help determine the best donor age and time in culture for optimal wound healing outcomes or more reproducible in vitro testing constructs.

Extracellular Matrix Hydrogels Promote Expression of Muscle-Tendon Junction Proteins.

Muscle and tendon injuries are prevalent and range from minor sprains and strains to traumatic, debilitating injuries. However, the interactions between these tissues during injury and recovery remain unclear. Three-dimensional tissue models that incorporate both tissues and a physiologically relevant junction between muscle and tendon may help understand how the two tissues interact. Here, we use tissue specific extracellular matrix (ECM) derived from muscle and tendon to determine how cells of each tissue interact with the microenvironment of the opposite tissue, resulting in junction-specific features. The ECM materials were derived from the Achilles tendon and gastrocnemius muscle, decellularized, and processed to form tissue-specific pre-hydrogel digests. The ECM materials were unique in respect to protein composition and included many types of ECM proteins, not just collagens. After digestion and gelation, ECM hydrogels had similar complex viscosities that were less than type I collagen hydrogels at the same concentration. C2C12 myoblasts and tendon fibroblasts were cultured in tissue-specific ECM conditioned media or encapsulated in tissue-specific ECM hydrogels to determine cell-matrix interactions and the effects on a muscle-tendon junction marker, paxillin. The ECM conditioned media had only a minor effect on the upregulation of paxillin in cells cultured in monolayer. However, cells cultured within ECM hydrogels had 50-70% higher paxillin expression than cells cultured in type I collagen hydrogels. Contraction of the ECM hydrogels varied by the type of ECM used. Subsequent experiments with a varying density of type I collagen (and thus contraction) showed no correlation between paxillin expression and the amount of gel contraction, suggesting that a constituent of the ECM was the driver of paxillin expression in the ECM hydrogels. In addition, another junction marker, type XXII collagen, had similar expression patterns as paxillin, with smaller effect sizes. Using tissue-specific ECM allowed for the de-construction of the cell-matrix interactions similar to muscle-tendon junctions to study the expression of myotendinous junction-specific proteins. Impact statement The muscle-tendon junction is an important feature of muscle-tendon units; however, despite crosstalk between the two tissue types, the junction is often overlooked in current research. Deconstructing the cell-matrix interactions will provide the opportunity to study significant junction-specific features and markers that should be included in tissue models of the muscle-tendon unit, while gaining a deeper understanding of the natural junction. This research aims at informing future methods to engineer a more relevant multi-tissue platform to study the muscle-tendon unit.

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.

Evaluation of a continuous locking novel epitendinous suture pattern with and without a core locking-loop suture on the biomechanical properties of tenorrhaphy constructs in an ex vivo model of canine superficial digital flexor tendon laceration.

OBJECTIVE: To evaluate the effect of a continuous locking novel epitendinous suture (nES) pattern with and without a core locking-loop (LL) suture on the biomechanical properties of ex vivo canine superficial digital flexor tendon (SDFT) tenorrhaphy constructs. SAMPLE: 54 cadaveric forelimb SDFTs from 27 musculoskeletally normal adult dogs. PROCEDURES: Tendons were assigned to 3 groups (18 SDFTs/group): sharply transected and repaired with a core LL suture alone (group 1), an nES pattern alone (group 2), or a combination of a core LL suture and nES pattern (group 3). All constructs underwent a single load-to-failure test. Yield, peak, and failure loads; gap formation incidence; and mode of failure were compared among the 3 groups. RESULTS: Mean yield, peak, and failure loads differed significantly among the 3 groups and were greatest for group 3 and lowest for group 1. Mean yield, peak, and failure loads for group 3 constructs were greater than those for group 1 constructs by 50%, 47%, and 44%, respectively. None of the group 3 constructs developed 3-mm gaps. The most common mode of failure was suture pulling through the tendon for groups 1 (12/18) and 2 (12/18) and suture breakage for group 3 (13/18). CONCLUSIONS AND CLINICAL RELEVANCE: Results suggested augmentation of a core LL suture with an nES pattern significantly increased the strength of and prevented 3-mm gap formation at the tenorrhaphy site in ex vivo canine SDFTs. In vivo studies are necessary to assess the effectiveness and practicality of the nES pattern for SDFT repair in dogs.

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.

Assessment of skin staples for augmentation of core tenorrhaphy in an ex vivo model of canine superficial digital flexor tendon laceration.

OBJECTIVE: To compare the biomechanical strength and incidence of gap formation among canine superficial digital flexor tendon (SDFT) constructs that underwent core tenorrhaphy only and those in which the core tenorrhaphy was augmented with skin staples or a continuous Silfverskiold cross-stitch (SXS) suture pattern. SAMPLE: 42 cadaveric forelimb SDFTs from 21 musculoskeletally normal dogs. PROCEDURES: Tendons were randomly assigned to 3 groups (14 SDTFs/group), sharply transected, and repaired with a core locking-loop suture alone (group 1) or augmented with circumferential placement of skin staples (group 2) or a continuous SXS suture pattern (group 3) in the epitenon. All constructs underwent a single load-to-failure test. Yield, peak, and failure loads, incidence of gap formation, and mode of failure were compared among the 3 groups. RESULTS: Mean yield, peak, and failure loads differed significantly among experimental groups and were greatest for group 3 and lowest for group 1 constructs. The incidence of gap formation differed among the tested groups and was lowest for group 3 and highest for group 1. The most common mode of construct failure was the suture pulling through the tendon for group 1, staple deformation for group 2, and epitendinous suture breakage for group 3. CONCLUSIONS AND CLINICAL RELEVANCE: Results indicated epitendinous placement of skin staples around a core SDFT tenorrhaphy site improved the biomechanical strength and resistance to gap formation for the repair but was inferior to epitendinous placement of SXS sutures. Further research is necessary before skin staples are used for tenorrhaphy augmentation in clinical patients.

Photoinduced reconfiguration to control the protein-binding affinity of azobenzene-cyclized peptides.

The impact of next-generation biorecognition elements (ligands) will be determined by the ability to remotely control their binding activity for a target biomolecule in complex environments. Compared to conventional mechanisms for regulating binding affinity (pH, ionic strength, or chaotropic agents), light provides higher accuracy and rapidity, and is particularly suited for labile targets. In this study, we demonstrate a general method to develop azobenzene-cyclized peptide ligands with light-controlled affinity for target proteins. Light triggers a cis/trans isomerization of the azobenzene, which results in a major structural rearrangement of the cyclic peptide from a non-binding to a binding configuration. Critical to this goal are the ability to achieve efficient photo-isomerization under low light dosage and the temporal stability of both cis and trans isomers. We demonstrated our method by designing photo-switchable peptides targeting vascular cell adhesion marker 1 (VCAM1), a cell marker implicated in stem cell function. Starting from a known VCAM1-binding linear peptide, an ensemble of azobenzene-cyclized variants with selective light-controlled binding were identified by combining in silico design with experimental characterization via spectroscopy and surface plasmon resonance. Variant cycloAZOB[G-VHAKQHRN-K] featured rapid, light-controlled binding of VCAM1 (KD,trans/KD,cis ∼ 130). Biotin-cycloAZOB[G-VHAKQHRN-K] was utilized to label brain microvascular endothelial cells (BMECs), showing co-localization with anti-VCAM1 antibodies in cis configuration and negligible binding in trans configuration.

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.

Fast Automated Approach for the Derivation of Acellular Extracellular Matrix Scaffolds from Porcine Soft Tissues.

Decellularized extracellular matrix (ECM) scaffolds derived from tissues and organs are complex biomaterials used in clinical and research applications. A number of decellularization protocols have been described for ECM biomaterials derivation, each adapted to a particular tissue and use, restricting comparisons among materials. One of the major sources of variability in ECM products comes from the tissue source and animal age. Although this variability could be minimized using established tissue sources, other sources arise from the decellularization process itself. Overall, current protocols require manual work and are poorly standardized with regard to the choice of reagents, the order by which they are added, and exposure times. The combination of these factors adds variability affecting the uniformity of the final product between batches. Furthermore, each protocol needs to be optimized for each tissue and tissue source making tissue-to-tissue comparisons difficult. Automation and standardization of ECM scaffold development constitute a significant improvement to current biomanufacturing techniques but remains poorly explored. This study aimed to develop a biofabrication method for fast and automated derivation of raw material for ECM hydrogel production while preserving ECM composition and controlling lot-to-lot variability. The main result was a closed semibatch bioreactor system with automated dosing of decellularization reagents capable of deriving ECM material from pretreated soft tissues. The ECM was further processed into hydrogels to demonstrate gelation and cytocompatibility. This work presents a versatile, scalable, and automated platform for the rapid production of ECM scaffolds.

Porcine Vocal Fold Lamina Propria-Derived Biomaterials Modulate TGF-ß1-Mediated Fibroblast Activation in Vitro.

The vocal fold lamina propria (VFLP), one of the outermost layers of the vocal fold (VF), is composed of tissue-specific extracellular matrix (ECM) proteins and is highly susceptible to injury. Various biomaterials have been clinically tested to treat voice disorders (e.g., hydrogels, fat, and hyaluronic acid), but satisfactory recovery of the VF functionality remains elusive. Fibrosis or scar formation in the VF is a major challenge, and the development and refinement of novel therapeutics that promote the healing and normal function of the VF are needed. Injectable hydrogels derived from native tissues have been previously reported with major advantages over synthetic hydrogels, including constructive tissue remodeling and reduced scar tissue formation. This study aims to characterize the composition of a decellularized porcine VFLP-ECM scaffold and the cytocompatibility and potential antifibrotic properties of a hydrogel derived from VFLP-ECM. In addition, we isolated potential matrix-bound vesicles (MBVs) and macromolecules from the VFLP-ECM that also downregulated smooth muscle actin ACTA2 under transforming growth factor-beta 1 (TGF-ß1) stimulation. The results provide evidence of the unique protein composition of the VFLP-ECM and the potential link between the components of the VFLP-ECM and the inhibition of TGF-ß1 signaling observed in vitro when transformed into injectable forms.

Effect of bite depth of an epitendinous suture on the biomechanical strength of repaired canine flexor tendons.

OBJECTIVE: To determine effects of bite depth for placement of an epitendinous suture on the biomechanical strength and gap formation of repaired canine tendons. SAMPLE: 48 superficial digital flexor tendons (SDFTs) obtained from 24 canine cadavers. PROCEDURES: Tendons were assigned to 3 groups (16 tendons/group). Each SDFT was transected and then repaired with a continuous epitendinous suture placed with a bite depth of 1, 2, or 3 mm for groups 1, 2, and 3, respectively. Specimens were loaded to failure. Failure mode, gap formation, yield force, peak force, and failure force were analyzed. RESULTS: Yield, peak, and failure forces differed significantly between groups 1 and 3 and groups 2 and 3 but not between groups 1 and 2. Comparison of the force resisted at 1 and 3 mm of gapping revealed a significant difference between groups 1 and 3 and groups 2 and 3 but not between groups 1 and 2. Failure mode did not differ among groups; suture pull-through occurred in 43 of 48 (89.6%) specimens. CONCLUSIONS AND CLINICAL RELEVANCE: Increasing bite depth of an epitendinous suture toward the center of the tendon substance increased repair site strength and decreased the incidence of gap formation. Repair of tendon injuries in dogs by use of an epitendinous suture with bites made deep into the tendon should result in a stronger repair, which potentially would allow loading and rehabilitation to begin sooner after surgery. Suture techniques should be investigated in vivo to determine effects on tendinous healing and blood supply before clinical implementation.

Platelet-like particles dynamically stiffen fibrin matrices and improve wound healing outcomes.

Native platelets perform several critical functions within the context of wound healing, including participating in initial hemostasis and interacting with fibrin at the wound site to induce clot retraction. Platelet depletion or dysfunction due to trauma or disease can inhibit robust wound healing responses. There has been a focus recently on developing synthetic, non-immunogenic platelet mimetic technologies for the purpose of augmenting hemostatic responses in cases of deficient native platelet functionality. Here we describe the application of synthetic platelet-like particles (PLPs), capable of recapitulating the deformable platelet body and fibrin specificity found in native platelets, to enhance healing outcomes. We first demonstrate PLPs mimic activated platelet morphology and induce fibrin clot retraction. During clot retraction, native platelets generate forces within a fibrin network to stiffen the fibrin matrix; therefore, we hypothesized that our PLPs will likewise be able to stiffen provisional fibrin matrices. Due to previous studies indicating that increased matrix stiffness is linked to increased cellular migration, we further hypothesize that PLP-mediated fibrin stiffening will enhance cell migration and improve healing outcomes within in vitro and in vivo models of wound healing. PLPs were found to enhance fibroblast migration in in vitro models of early wound healing and enhance healing outcomes in an in vivo murine model of wound healing. These studies demonstrate the utility of PLPs for enhancing wound repair and also provide insight into the role of native platelet-mediated clot retraction in wound healing.

Macrophages' Role in Tissue Disease and Regeneration.

Inflammation is an essential component of the normal mammalian host tissue response and plays an important role during cardiovascular and musculoskeletal diseases. Given the important role of inflammation on the host tissue response after injury, understanding this process represents essential aspects of biomedical research, tissue engineering, and regenerative medicine. Macrophages are central players during the inflammatory response with an extensive role during wound healing. These cells exhibit a spectrum of activation states that span from pro-inflammatory to pro-healing phenotypes. The phenotype of the macrophages can have profound influences on the progression of disease or injury. As such, understanding and subsequent modulation of macrophage phenotype represents an exciting target area for regenerative medicine therapies. In this chapter, we describe the role of macrophages in specific cases of injury and disease. After myocardial infarction, a biphasic response of pro- and anti-inflammatory macrophages are involved in the remodeling process. In volumetric muscle loss, there is an intricate communication between inflammatory cells and progenitor cells affecting repair processes. Osteoarthritis is characterized by increased levels of pro-inflammatory macrophages over an extended period of time with significant impact on the progression of the disease. By harnessing the complex role of macrophages, enhanced therapeutic treatments can be developed that enhance the normal healing response as well as help the survival of therapeutic cells delivered to the site of injury.