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Objective We have recently shown that mesenchymal stem cells (MSCs) embedded in a hyaluronic acid (HA) hydrogel and exposed to chondrogenic factors (transforming growth factor–β3 [TGF-β3]) produce a cartilage-like tissue in vitro. The current objective was to determine if these same factors could be combined immediately prior to implantation to induce a superior healing response in vivo relative to the hydrogel alone. Design Trochlear chondral defects were created in Yucatan mini-pigs (6 months old). Treatment groups included an HA hydrogel alone and hydrogels containing allogeneic MSCs, TGF-β3, or both. Six weeks after surgery, micro-computed tomography was used to quantitatively assess defect fill and subchondral bone remodeling. The quality of cartilage repair was assessed using the ICRS-II histological scoring system and immunohistochemistry for type II collagen. Results Treatment with TGF-β3 led to a marked increase in positive staining for collagen type II within defects ( P < 0.05), while delivery of MSCs did not ( P > 0.05). Neither condition had an impact on other histological semiquantitative scores ( P > 0.05), and inclusion of MSCs led to significantly less defect fill ( P < 0.05). For all measurements, no synergistic interaction was found between TGF-β3 and MSC treatment when they were delivered together ( P > 0.05). Conclusions At this early healing time point, treatment with TGF-β3 promoted the formation of collagen type II within the defect, while allogeneic MSCs had little benefit. Combination of TGF-β3 and MSCs at the time of surgery did not produce a synergistic effect. An in vitro precultured construct made of these components may be required to enhance in vivo repair in this model system.

In this work, a chemomechanical model describing the growth dynamics of cell-matrix adhesion structures (i.e., focal adhesions (FAs)) is developed. We show that there are three regimes for FA evolution depending on their size. Specifically, nascent adhesions with initial lengths below a critical value that are yet to engage in actin fibers will dissolve, whereas bigger ones will grow into mature FAs with a steady state size. In adhesions where growth surpasses the steady state size, disassembly will occur until their sizes are reduced to the equilibrium state. This finding arises from the fact that polymerization of adhesion proteins is force-dependent. Under actomyosin contraction, individual integrin bonds within small FAs (i.e., nascent adhesions or focal complexes) must transmit higher loads while the phenomenon of stress concentration occurs at the edge of large adhesion patches. As such, an effective stiffness of the FA-extracellular matrix complex that is either too small or too large will be relatively low, resulting in a limited actomyosin pulling force developed at the edge that is insufficient to prevent disassembly. Furthermore, it is found that a stiffer extracellular matrix and/or nucleus, as well as a stronger chemomechanical feedback, will induce larger adhesions along with a higher level of contraction force. Interestingly, switching the extracellular side from an elastic half-space, corresponding to some widely used in vitro gel substrates, to a one-dimensional fiber (as in the case of cells anchoring to a fibrous scaffold in vivo) does not qualitative change these conclusions. Our model predictions are in good agreement with a variety of experimental observations obtained in this study as well as those reported in the literature. Furthermore, this new model, to our knowledge, provides a framework with which to understand how both intracellular and extracellular perturbations lead to changes in adhesion structure number and size.

Objectives/Hypothesis To develop an effective rabbit model of in vitro- and in vivo-derived tissue-engineered cartilage for laryngotracheal reconstruction (LTR). Study Design 1) Determination of the optimal scaffold 1% hyaluronic acid (HA), 2% HA, and polyglycolic acid (PGA) and in vitro culture time course using a pilot study of 4 by 4-mm in vitro-derived constructs analyzed on a static culture versus zero-gravity bioreactor for 4, 8, and 12 weeks, with determination of compressive modulus and histology as outcome measures. 2) Three-stage survival rabbit experiment utilizing autologous auricular chondrocytes seeded in scaffolds, either 1% HA or PGA. The constructs were cultured for the determined in vitro time period and then cultured in vivo for 12 weeks. Fifteen LTRs were performed using HA cartilage constructs, and one was performed with a PGA construct. All remaining specimens and the final reconstructed larynx underwent mechanical testing, histology, and glycosaminoglycan (GAG) content determination, and then were compared to cricoid control specimens (n = 13) and control LTR using autologous thyroid cartilage (n = 18). Methods 1) One rabbit underwent an auricular punch biopsy, and its chondrocytes were isolated and expanded and then encapsulated in eight 4 by 4-mm discs of 1% HA, 2% HA, PGA either in rotary bioreactor or static culture for 4, 8, and 12 weeks, respectively, with determination of compressive modulus, GAG content, and histology. 2) Sixteen rabbits underwent ear punch biopsy; chondrocytes were isolated and expanded. The cells were seeded in 13 by 5 by 2.25-mm UV photopolymerized 1% HA (w/w) or calcium alginate encapsulated synthetic PGA (13 × 5 × 2 mm); the constructs were then incubated in vitro for 12 weeks (the optimal time period determined above in paragraph 1) on a shaker. One HA and one PGA construct from each animal was tested mechanically and histologically, and the remaining eight (4 HA and 4 PGA) were implanted in the neck. After 12 weeks in vivo, the most optimal-appearing HA construct was used as a graft for LTR in 15 rabbits and PGA in one rabbit. The seven remaining specimens underwent hematoxylin and eosin, Safranin O, GAG content determination, and flexural modulus testing. At 12 weeks postoperative, the animals were euthanized and underwent endoscopy. The larynges underwent mechanical and histological testing. All animals that died underwent postmortem examination, including gross and microhistological analysis of the reconstructed airway. Results Thirteen of the 15 rabbits that underwent LTR with HA in vitro- and in vivo-derived tissue-engineered cartilage constructs survived. The 1% HA specimens had the highest modulus and GAG after 12 weeks in vitro. The HA constructs became well integrated in the airway, supported respiration for the 12 weeks, and were histologically and mechanically similar to autologous cartilage. Conclusions The engineering of in vitro- and in vivo-derived cartilage with HA is a novel approach for laryngotracheal reconstruction. The data suggests that the in vitro- and in vivo-derived tissue-engineered approaches may offer a promising alternative to current strategies used in pediatric airway reconstruction, as well as other head and neck applications. Level of Evidence NA. Laryngoscope, 2015

Supraspinatus tears often result in the setting of chronic tendinopathy. However, the typical repair model utilizes an acute injury. In recognition of that distinction, our laboratory developed an overuse animal model; however it is unclear whether induced overuse is necessary in the repair model. We studied the repair properties of overuse-induced tendons compared to normal tendons. We hypothesized that histological and mechanical properties would not be altered between the overuse-induced and normal tendons 1 and 4 weeks after repair. Thirty-one adult male Sprague-Dawley rats were subjected to either overuse or cage activity for 4 weeks prior to bilateral supraspinatus tendon repair surgery. Rats were sacrificed at 1 and 4 weeks post-surgery and evaluated for histology and mechanics. Results at 1 week showed no clear histologic changes, but increased inflammatory protein expression in overuse tendons. At 4 weeks, percent relaxation was slightly increased in the overuse group. No other alterations in mechanics or histology were observed. Our results suggest that the effects of the surgical injury overshadow the changes evoked by overuse. Because clinically relevant mechanical parameters were not altered in the overuse group, we conclude that when examining tendons 4 weeks after repair in the classic rat supraspinatus model, inducing overuse prior to surgery is likely to be unnecessary.

Degeneration of the intervertebral discs is strongly implicated as a cause of low back pain. Since current treatments for discogenic low back pain show poor long-term efficacy, a number of new biological strategies are being pursued. For such therapies to succeed, it is critical that they be validated in conditions that mimic the unique biochemical microenvironment of the nucleus pulposus (NP), which include low oxygen tension. Therefore, the objective of this study was to investigate the effects of oxygen tension on NP cell functional extracellular matrix elaboration in 3D culture. Bovine NP cells were encapsulated in agarose constructs and cultured for 14 or 42 days in either 20% or 2% oxygen in defined media containing transforming growth factor beta-3. At each time point, extracellular matrix composition, biomechanics, and mRNA expression of key phenotypic markers were evaluated. Results showed that while bulk mechanics and composition were largely independent of oxygen level, low oxygen promoted improved restoration of the NP phenotype, higher mRNA expression of extracellular matrix and NP specific markers, and more uniform matrix elaboration. These findings indicate that culture under physiological oxygen levels is an important consideration for successful development of cell and growth factor-based regenerative strategies for the disc.

When the field of tissue engineering first arose, scaffolds were conceived of as inert three-dimensional structures whose primary function was to support cellularity and tissue growth. Since then, advances in scaffold and biomaterial design have evolved to not only guide tissue formation, but also to interact dynamically with and manipulate the wound environment. At present, these efforts are being directed towards strategies that directly address limitations in endogenous wound repair, with the goal of reprogramming the local wound environment (and the cells within that locality) from a state that culminates in an inferior tissue repair into a state in which functional regeneration is achieved. This review will address this approach with a focus on recent advances in scaffold design towards the resolution of tears of the knee meniscus as a case example. The inherent limitations to endogenous repair will be discussed, as will specific examples of how biomaterials are being designed to overcome these limitations. Examples will include design of fibrous scaffolds that promote colonization by modulating local extracellular matrix density and delivering recruitment factors. Furthermore, we will discuss scaffolds that are themselves modulated by the wound environment to alter porosity and modulate therapeutic release through precise coordination of scaffold degradation. Finally, we will close with emerging concepts in local control of cell mechanics to improve interstitial cell migration and so advance repair. Overall, these examples will illustrate how emergent features within a biomaterial can be tuned to manipulate and harness the local tissue microenvironment in order to promote robust regeneration.

Preclinical large animal models are essential for evaluating new tissue engineering (TE) technologies and refining surgical approaches for cartilage repair. Some preclinical animal studies, including the commonly used minipig model, have noted marked remodeling of the subchondral bone. However, the mechanisms underlying this response have not been well characterized. Thus, our objective was to compare in-vivo outcomes of chondral defects with varied injury depths and treatments.Trochlear chondral defects were created in 11 Yucatan minipigs (6 months old). Groups included an untreated partial-thickness defect (PTD), an untreated full-thickness defect (FTD), and FTDs treated with microfracture, autologous cartilage transfer (FTD-ACT), or an acellular hyaluronic acid hydrogel. Six weeks after surgery, micro-computed tomography (μCT) was used to quantitatively assess defect fill and subchondral bone remodeling. The quality of cartilage repair was assessed using the ICRS-II histological scoring system and immunohistochemistry for type II collagen. A finite element model (FEM) was developed to assess load transmission.Using μCT, substantial bone remodeling was observed for all FTDs, but not for the PTD group. The best overall histological scores and greatest type II collagen staining was found for the FTD-ACT and PTD groups. The FEM confirmed that only the FTD-ACT group could initially restore appropriate transfer of compressive loads to the underlying bone.The bony remodeling observed in this model system appears to be a biological phenomena and not a result of altered mechanical loading, with the depth of the focal chondral defect (partial vs. full thickness) dictating the bony remodeling response. The type of cartilage injury should be carefully controlled in studies utilizing this model to evaluate TE approaches for cartilage repair.

The transmembrane glycoprotein N-cadherin (NCad) mediates cell–cell interactions found during mesenchymal condensation and chondrogenesis. Here, NCad-derived peptides (i.e., HAV) were incorporated into hyaluronic acid (HA) hydrogels with encapsulated mesenchymal stem cells (MSCs). Since the dose and timing of NCad signaling are dynamic, the presentation of HAV peptide presentation was tuned via alterations in peptide concentration and incorporation of an ADAM10-cleavable domain between the hydrogel and the HAV motif, respectively. HA hydrogels functionalized with HAV resulted in dose-dependent increases in early chondrogenesis of encapsulated MSCs and in resultant cartilage matrix production. For example, type II collagen and glycosaminoglycan production increased ~9- and 2-fold with the highest dose of HAV (i.e., 2 mM), respectively, when compared to unmodified hydrogels, while incorporation of an efficient ADAM10-cleavable domain between the HAV peptide and hydrogel abolished increases in chondrogenesis and matrix production. Treatment with a small-molecule ADAM10 inhibitor restored the functional effect of the HAV peptide, indicating that timing and duration of HAV peptide presentation is crucial for robust chondrogenesis. This study demonstrates a nuanced approach to the biofunctionalization of hydrogels to better emulate the complex cell microenvironment during embryogenesis towards stem cell-based cartilage production.

Degradation of the nucleus pulposus (NP) is an early hallmark of intervertebral disc degeneration. The capacity for endogenous regeneration in the NP is limited due to the low cellularity and poor nutrient supply of this avascular tissue. Towards restoring the NP, a number of biomaterials have been explored for cell delivery. These materials must support the NP cell phenotype while promoting the elaboration of an NP-like extracellular matrix in the shortest possible time. Our previous work with chondrocytes and mesenchymal stem cells demonstrated that hydrogels based on hyaluronic acid (HA) are effective at promoting matrix production and the development of functional material properties. However, this material has not been evaluated in the context of NP cells. Therefore, to test this material for NP regeneration, bovine NP cells were encapsulated in 1% w/vol HA hydrogels at either a low seeding density (20 × 106 cells/ml) or a high seeding density (60 × 106 cells/ml), and constructs were cultured over an 8 week period. These engineered NP cell-laden HA hydrogels showed functional matrix accumulation, with increasing matrix content and mechanical properties with time in culture at both seeding densities. Furthermore, encapsulated cells showed NP-specific gene expression profiles that were significantly higher than expanded NP cells prior to encapsulation, suggesting a restoration of phenotype. Interestingly, these levels were higher at the lower seeding density compared to the higher seeding density. These findings support the use of HA-based hydrogels for NP tissue engineering and cellular therapies directed at restoration or replacement of the endogenous NP.

Mesenchymal stem cells (MSCs) hold great promise for regenerative therapies and tissue engineering applications given their multipotential differentiation capacity. However, MSC isolation and expansion are typically performed on super-physiologically stiff tissue culture plastic (TCP), which may alter their behavior and lead to unintended consequences upon implantation. In contrast, electrospun nanofibrous scaffolds possess physical and mechanical properties that are similar to that of native tissue. In this study, we investigated whether isolation and expansion of juvenile bovine MSCs directly onto electrospun nanofibrous scaffolds better preserves MSC phenotype and stemness compared to TCP. Our data show that culture of MSCs on electrospun scaffolds reduces proliferation, decreases cellular senescence, and better maintains stemness compared to cells isolated and expanded on TCP, likely due to a reduction in cell contractility. Furthermore, in contrast to electrospun scaffolds, TCP biased MSCs towards a fibrotic phenotype that persisted even after the cells were reseeded onto a different substrate. Cells pre-cultured on electrospun scaffolds exhibited a heightened response to mechanical stimuli and greater chondrogenesis in methacrylated hyaluronic acid hydrogels. These data suggest that alternative substrates that better approximate the native cell environment should be used to preserve endogenous MSC behavior and may improve their success in tissue engineering applications. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:808-815, 2018.

Replacement of the intervertebral disc with a viable, tissue-engineered construct that mimics native tissue structure and function is an attractive alternative to fusion or mechanical arthroplasty for the treatment of disc pathology. While a number of engineered discs have been developed, the average size of these constructs remains a fraction of the size of human intervertebral discs. In this study, we fabricated medium (3 mm height × 10 mm diameter) and large (6 mm height × 20 mm diameter) sized disc-like angle ply structures (DAPS), encompassing size scales from the rabbit lumbar spine to the human cervical spine. Maturation of these engineered discs was evaluated over 15 weeks in culture by quantifying cell viability and metabolic activity, construct biochemical content, MRI T2 values, and mechanical properties. To assess the performance of the DAPS in the in vivo space, pre-cultured DAPS were implanted subcutaneously in athymic rats for 5 weeks. Our findings show that both sized DAPS matured functionally and compositionally during in vitro culture, as evidenced by increases in mechanical properties and biochemical content over time, yet large DAPS under-performed compared to medium DAPS. Subcutaneous implantation resulted in reductions in NP cell viability and GAG content at both size scales, with little effect on AF biochemistry or metabolic activity. These findings demonstrate that engineered discs at large size scales will mature during in vitro culture, however, future work will need to address the challenges of reduced cell viability and heterogeneous matrix distribution throughout the construct. Statement of Significance This work establishes, for the first time, tissue-engineered intervertebral discs for total disc replacement at large, clinically relevant length scales. Clinical translation of tissue-engineered discs will offer an alternative to mechanical disc arthroplasty and fusion procedures, and may contribute to a paradigm shift in the clinical care for patients with disc pathology and associated axial spine and neurogenic extremity pain.

Dense connective tissue injuries have limited repair, due to the paucity of cells at the wound site. We hypothesize that decreasing the density of the local extracellular matrix (ECM) in conjunction with releasing chemoattractive signals increases cellularity and tissue formation after injury. Using the knee meniscus as a model system, we query interstitial cell migration in the context of migratory barriers using a novel tissue Boyden chamber and show that a gradient of platelet-derived growth factor-AB (PDGF-AB) expedites migration through native tissue. To implement these signals in situ, we develop nanofibrous scaffolds with distinct fiber fractions that sequentially release active collagenase (to increase ECM porosity) and PDGF-AB (to attract endogenous cells) in a localized and coordinated manner. We show that, when placed into a meniscal defect, the controlled release of collagenase and PDGF-AB increases cellularity at the interface and within the scaffold, as well as integration with the surrounding tissue., Dense connective tissues do not easily heal, in part due to a low supply of reparative cells. Here, the authors develop a fibrous scaffold for meniscal repair that sequentially releases collagenase and a growth factor at the injury site, breaking down the extracellular matrix and recruiting endogenous cells.

The development of engineered tissues has progressed over the past 20 years from in vitro characterization to in vivo implementation. For musculoskeletal tissue engineering in particular, the emphasis of many of these studies was to select conditions that maximized functional and compositional gains in vitro. However, the transition from the favorable in vitro culture environment to a less favorable in vivo environment has proven difficult, and, in many cases, engineered tissues do not retain their preimplantation phenotype after even short periods in vivo. Our laboratory recently developed disc-like angle-ply structures (DAPS), an engineered intervertebral disc for total disc replacement. In this study, we tested six different preculture media formulations (three serum-containing and three chemically defined, with varying doses of transforming growth factor β3 [TGF-β3] and varying strategies to introduce serum) for their ability to preserve DAPS composition and metabolic activity during the transition from in vitro culture to in vivo implantation in a subcutaneous athymic rat model. We assayed implants before and after implantation to determine collagen content, glycosaminoglycan (GAG) content, metabolic activity, and magnetic resonance imaging (MRI) characteristics. A chemically defined media condition that incorporated TGF-β3 promoted the deposition of GAG and collagen in DAPS in vitro, the maintenance of accumulated matrix in vivo, and minimal changes in the metabolic activity of cells within the construct. Preculture in serum-containing media (with or without TGF-β3) was not compatible with DAPS maturation, particularly in the nucleus pulposus (NP) region. All groups showed increased collagen production after implantation. These findings define a favorable preculture strategy for the translation of engineered discs seeded with disc cells.

The purpose of study was to investigate the maturation of mesenchymal stem cells (MSC) laden in HA constructs with various combinations of chemically defined medium (CM) components and determine the impact of dexamethasone and serum on construct properties. Constructs were cultured in CM with the addition or withdrawal of media components or were transferred to serum containing media that partially represents an in vivo-like condition where pro-inflammatory signals are present. Constructs cultured in CM+ (CM with TGF-β3) and DEX- (CM+ without dexamethasone) conditions produced robust matrix, while those in ITS/BSA/LA- (CM+ without ITS/BSA/LA) and Serum+ (10% FBS with TGF-β3) produced little matrix. While construct properties in DEX- were greater than those in CM+ at 4 weeks, properties in CM+ and DEX- reversed by 8 weeks. While construct properties in DEX- were greater than those in CM+ at 4 weeks, the continued absence or removal of dexamethasone resulted in marked GAG loss by 8 weeks. Conversely, the continued presence or new addition of dexamethasone at 4 weeks further improved or maintained construct properties through 8 weeks. Finally, when constructs were converted to Serum (in the continued presence of TGF-β3 with or without dexamethasone) after pre-culture in CM+ for 4 weeks, GAG loss was attenuated with addition of dexamethasone. Interestingly, however, collagen content and type was not impacted. In conclusion, dexamethasone influences the functional maturation of MSC-laden HA constructs, and may help to maintain properties during long-term culture or with in vivo translation by repressing pro-inflammatory signals. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:1717-1727, 2018.

Cartilage tissue engineering is emerging as a promising treatment for osteoarthritis, and the field has progressed toward utilizing large animal models for proof of concept and preclinical studies. Mechanical testing of the regenerative tissue is an essential outcome for functional evaluation. However, testing modalities and constitutive frameworks used to evaluate in vitro grown samples differ substantially from those used to evaluate in vivo derived samples. To address this, we developed finite element (FE) models (using FEBio) of unconfined compression and indentation testing, modalities commonly used for such samples. We determined the model sensitivity to tissue radius and subchondral bone modulus, as well as its ability to estimate material parameters using the built-in parameter optimization tool in FEBio. We then sequentially tested agarose gels of 4%, 6%, 8%, and 10% weight/weight using a custom indentation platform, followed by unconfined compression. Similarly, we evaluated the ability of the model to generate material parameters for living constructs by evaluating engineered cartilage. Juvenile bovine mesenchymal stem cells were seeded (2 × 107 cells/mL) in 1% weight/volume hyaluronic acid hydrogels and cultured in a chondrogenic medium for 3, 6, and 9 weeks. Samples were planed and tested sequentially in indentation and unconfined compression. The model successfully completed parameter optimization routines for each testing modality for both acellular and cell-based constructs. Traditional outcome measures and the FE-derived outcomes showed significant changes in material properties during the maturation of engineered cartilage tissue, capturing dynamic changes in functional tissue mechanics. These outcomes were significantly correlated with one another, establishing this FE modeling approach as a singular method for the evaluation of functional engineered and native tissue regeneration, both in vitro and in vivo.

Injury to the meniscus is common, but few viable strategies exist for its repair or regeneration. To address this, animal models have been developed to translate new treatment strategies toward the clinic. However, there is not yet a regulatory document guiding such studies. The purpose of this study was to carry out a systematic review of the literature on meniscus treatment methods and outcomes to define the state of the field. Public databases were queried by using search terms related to animal models and meniscus injury and/or repair over the years 1980-2015. Identified peer-reviewed manuscripts were screened by using Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. One of nine reviewers read each manuscript and scored them based on whether the publication described a series of predefined study descriptors and outcome measures. Additional data were extracted to identify common assays used. A total of 128 full-length peer-reviewed manuscripts were identified. The number of publications increased over the time frame analyzed, with 48% focused on augmented repair. Rabbit was, by far, the most prevalent species utilized (46%), with dog (21%) and sheep (20%) being the next most common. Analysis of study descriptors revealed that most studies appropriately documented details of the animal used, the surgical approach, and defect and implant characteristics (e.g., 63% of studies identified clearly the defect size). In terms of outcome parameters, most studies carried out macroscopic (85%), histologic (90%), and healing/integration (83%) analyses of the meniscus. However, many studies did not provide further analysis beyond these fundamental measures, and less than 40% reported on the adjacent cartilage and synovium, as well as joint function. There is intense interest in the field of meniscus repair. However, given the current lack of guidance documentation in this area, preclinical animal models are not performed in a standardized fashion. The development of a "Best Practices" document would increase reproducibility and external validity of experiments, while accelerating advancements in translational research. Advancement is of paramount importance given the high prevalence of meniscal injuries and the paucity of effective repair or regenerative strategies.

Hyaluronic acid (HA) has been widely used for cartilage tissue engineering applications. However, the optimal time point to harvest HA-based engineered constructs for cartilage repair is still under investigation. In this study, we investigated the ability of a nondestructive modality, near-infrared spectroscopic (NIR) analysis, to predict compositional and mechanical properties of HA-based engineered cartilage constructs. NIR spectral data were collected from control, unseeded constructs, and twice per week by fiber optic from constructs seeded with chondrocytes during their development over an 8-week period. Constructs were harvested at 2, 4, 6, and 8 weeks, collagen and sulfated glycosaminoglycan content measured using biochemical assays, and the mechanical properties of the constructs evaluated using unconfined compression tests. NIR absorbances associated with the scaffold material, water, and engineered cartilage matrix, were identified. The NIR-determined matrix absorbance plateaued after 4 weeks of culture, which was in agreement with the biochemical assay results. Similarly, the mechanical properties of the constructs also plateaued at 4 weeks. A multivariate partial least square model based on NIR spectral input was developed to predict the moduli of the constructs, which resulted in a prediction error of 10% and R value of 0.88 for predicted versus actual values of dynamic modulus. Furthermore, the maximum increase in moduli was calculated from the first derivative of the curve fit of NIR-predicted and actual moduli values over time, and both occurred at ∼2 weeks. Collectively, these data suggest that NIR spectral data analysis could be an alternative to destructive biochemical and mechanical methods for evaluation of HA-based engineered cartilage construct properties.

Objective To test different fixation methods of a 3-dimensionally woven poly(ϵ-caprolactone) (PCL) scaffold within chondral defects of a weightbearing large animal model. Methods Full thickness chondral defects were made in the femoral condyles of 15 adult male Yucatan mini-pigs. Two surgical approaches were compared including total arthrotomy (traditional) and a retinaculum-sparing, minimally invasive surgery (MIS) approach. Following microfracture (MFX), scaffolds were placed without fixation or were fixed with fibrin glue, suture, or subchondral anchor. Experimental endpoints were between 1 and 6 weeks. Micro–computed tomography and histology were used to assess samples. Results The MIS approach was superior as the traditional approach caused medial condyle cartilage wear. One of 13 (7.7%) of scaffolds without fixation, 4 of 11 (36.3%) fibrin scaffolds, 1 of 4 (25%) of sutured scaffolds, and 9 of 9 (100%) of anchor-fixed scaffolds remained in place. Histology demonstrated tissue filling with some overgrowth of PCL scaffolds. Conclusions Of the methods tested, the MIS approach coupled with subchondral anchor fixation provided the best scaffold retention in a mini-pig chondral defect model. This finding has implications for fixation strategies in future animal studies and potential future human use.

To understand how the complex biomechanical functions of the meniscus are endowed by the nanostructure of its extracellular matrix (ECM), we studied the anisotropy and heterogeneity in the micromechanical properties of the meniscus ECM. We used atomic force microscopy (AFM) to quantify the time-dependent mechanical properties of juvenile bovine meniscus at deformation length scales corresponding to the diameters of collagen fibrils. At this scale, anisotropy in the elastic modulus of the circumferential fibers, the major ECM structural unit, can be attributed to differences in fibril deformation modes: uncrimping when normal to the fiber axis, and laterally constrained compression when parallel to the fiber axis. Heterogeneity among different structural units is mainly associated with their variations in microscale fiber orientation, while heterogeneity across anatomical zones is due to alterations in collagen fibril diameter and alignment at the nanoscale. Unlike the elastic modulus, the time-dependent properties are more homogeneous and isotropic throughout the ECM. These results enable a detailed understanding of the meniscus structure-mechanics at the nanoscale, and can serve as a benchmark for understanding meniscus biomechanical functions, documenting disease progression and designing tissue repair strategies. Statement of Significance Meniscal damage is a common cause of joint injury, which can lead to the development of post-traumatic osteoarthritis among young adults. Restoration of meniscus function requires repairing its highly heterogeneous and complex extracellular matrix. Employing AFM, this study quantifies the anisotropic and heterogeneous features of the meniscus ECM structure and mechanics. The micromechanical properties are interpreted within the context of the collagen fibril nanostructure and its variation with tissue anatomical locations. These results provide a fundamental structure-mechanics knowledge benchmark, against which, repair and regeneration strategies can be developed and evaluated with respect to the specialized structural and functional complexity of the native tissue.

The meniscus is comprised of circumferentially aligned fibers that resist the tensile forces within the meniscus (i.e., hoop stress) that develop during loading of the knee. Although these circumferential fibers are severed by radial meniscal tears, tibial contact stresses do not increase until the tear reaches ∼90% of the meniscus width, suggesting that the severed circumferential fibers still bear load and maintain the mechanical functionality of the meniscus. Recent data demonstrates that the interfibrillar matrix can transfer strain energy to disconnected fibrils in tendon fascicles. In the meniscus, interdigitating radial tie fibers, which function to stabilize and bind the circumferential fibers together, are hypothesized to function in a similar manner by transmitting load to severed circumferential fibers near a radial tear. To test this hypothesis, we developed an engineered fibrous analog of the knee meniscus using poly(e-caprolactone) to create aligned scaffolds with variable amounts of non-aligned elements embedded within the scaffold. We show that the tensile properties of these scaffolds are a function of the ratio of aligned to non-aligned elements, and change in a predictable fashion following a simple mixture model. When measuring the loss of mechanical function in scaffolds with a radial tear, compared to intact scaffolds, the decrease in apparent linear modulus was reduced in scaffolds containing non-aligned layers compared to purely aligned scaffolds. Increased strains in areas adjacent to the defect were also noted in composite scaffolds. These findings indicate that non-aligned (disorganized) elements interspersed within an aligned network can improve overall mechanical function by promoting strain transfer to nearby disconnected fibers. This finding supports the notion that radial tie fibers may similarly promote tear tolerance in the knee meniscus, and will direct changes in clinical practice and provide guidance for tissue engineering strategies. Statement of Significance The meniscus is a complex fibrous tissue, whose architecture includes radial tie fibers that run perpendicular to and interdigitate with the predominant circumferential fibers. We hypothesized that these radial elements function to preserve mechanical function in the context of interruption of circumferential bundles, as would be the case in a meniscal tear. To test this hypothesis, we developed a biomaterial analog containing disorganized layers enmeshed regularly throughout an otherwise aligned network. Using this material formulation, we showed that strain transmission is improved in the vicinity of defects when disorganized fiber layers were present. This supports the idea that radial elements within the meniscus improve function near a tear, and will guide future clinical interventions and the development of engineered replacements.

Magnetic resonance imaging (MRI) with T2-weighting is routinely performed to assess intervertebral disc degeneration. Standard clinical evaluations of MR images are qualitative, however, and do not focus on region-specific alterations in the disc. Utilizing a rabbit needle puncture model, T2 mapping was performed on injured discs to develop a quantitative description of the degenerative process following puncture. To do so, an 18G needle was inserted into four discs per rabbit (L3/L4 to L6/L7) and T2 maps were generated pre- and 4 weeks post-injury. Individual T2 maps were normalized to a disc-specific coordinate system and then averaged for pre- and post-injury population composite T2 maps. We also developed a method to automatically segment the nucleus pulposus by fitting the NP region of the T2 maps with modified 2-D and 3-D Gaussian distribution functions. Puncture injury produced alterations in MR signal intensity in a region-specific manner mirroring human degeneration. Population average T2 maps provided a quantitative representation of the injury response, and identified deviations of individual degenerate discs from the pre-injury population. We found that the response to standardized injury was modest at lower lumbar levels, likely as a result of increased disc dimensions. These tools will be valuable for the quantitative characterization of disc degeneration in future clinical and pre-clinical studies. © 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 33:140–148, 2015.

Summary Objective A number of in vitro models of post-traumatic osteoarthritis (PTOA) have been developed to study the effect of mechanical overload on the processes that regulate cartilage degeneration. While such frameworks are critical for the identification therapeutic targets, existing technologies are limited in their throughput capacity. Here, we validate a test platform for high-throughput mechanical injury incorporating engineered cartilage. Method We utilized a high-throughput mechanical testing platform to apply injurious compression to engineered cartilage and determined their strain and strain rate dependent responses to injury. Next, we validated this response by applying the same injury conditions to cartilage explants. Finally, we conducted a pilot screen of putative PTOA therapeutic compounds. Results Engineered cartilage response to injury was strain dependent, with a 2-fold increase in glycosaminoglycan (GAG) loss at 75% compared to 50% strain. Extensive cell death was observed adjacent to fissures, with membrane rupture corroborated by marked increases in lactate dehydrogenase (LDH) release. Testing of established PTOA therapeutics showed that pan-caspase inhibitor [Z-VAD-FMK (ZVF)] was effective at reducing cell death, while the amphiphilic polymer [Poloxamer 188 (P188)] and the free-radical scavenger [ N -Acetyl-L-cysteine (NAC)] reduced GAG loss as compared to injury alone. Conclusions The injury response in this engineered cartilage model replicated key features of the response of cartilage explants, validating this system for application of physiologically relevant injurious compression. This study establishes a novel tool for the discovery of mechanisms governing cartilage injury, as well as a screening platform for the identification of new molecules for the treatment of PTOA.

Current clinical treatment after tendon repairs often includes prescribing NSAIDs to limit pain and inflammation. The negative influence of NSAIDs on bone repair is well documented, but their effects on tendon healing are less clear. While NSAIDs may be detrimental to early tendon healing, some evidence suggests that they may improve healing if administered later in the repair process.We asked whether the biomechanical and histologic effects of systemic ibuprofen administration on tendon healing are influenced by either immediate or delayed drug administration.After bilateral supraspinatus detachment and repair surgeries, rats were divided into groups and given ibuprofen orally for either Days 0 to 7 (early) or Days 8 to 14 (delayed) after surgery; a control group did not receive ibuprofen. Healing was evaluated at 1, 2, and 4 weeks postsurgery through biomechanical testing and histologic assessment.Biomechanical evaluation resulted in decreased stiffness and modulus at 4 weeks postsurgery for early ibuprofen delivery (mean ± SD [95% CI]: 10.8 ± 6.4 N/mm [6.7-14.8] and 8.9 ± 5.9 MPa [5.4-12.3]) when compared to control repair (20.4 ± 8.6 N/mm [16.3-24.5] and 15.7 ± 7.5 MPa [12.3-19.2]) (p = 0.003 and 0.013); however, there were no differences between the delayed ibuprofen group (18.1 ± 7.4 N/mm [14.2-22.1] and 11.5 ± 5.6 MPa [8.2-14.9]) and the control group. Histology confirmed mechanical results with reduced fiber reorganization over time in the early ibuprofen group.Early administration of ibuprofen in the postoperative period was detrimental to tendon healing, while delayed administration did not affect tendon healing.Historically, clinicians have often prescribed ibuprofen after tendon repair, but this study suggests that the timing of ibuprofen administration is critical to adequate tendon healing. This research necessitates future clinical studies investigating the use of ibuprofen for pain control after rotator cuff repair and other tendon injuries.

Intervertebral disc degeneration is implicated as a major cause of low-back pain. There is a pressing need for new regenerative therapies for disc degeneration that restore native tissue structure and mechanical function. To that end we investigated the therapeutic potential of an injectable, triple-interpenetrating-network hydrogel comprised of dextran, chitosan, and teleostean, for functional regeneration of the nucleus pulposus (NP) of the intervertebral disc in a series of biomechanical, cytotoxicity, and tissue engineering studies. Biomechanical properties were evaluated as a function of gelation time, with the hydrogel reaching ∼90% of steady-state aggregate modulus within 10 h. Hydrogel mechanical properties evaluated in confined and unconfined compression were comparable to native human NP properties. To confirm containment within the disc under physiological loading, toluidine-blue-labeled hydrogel was injected into human cadaveric spine segments after creation of a nucleotomy defect, and the segments were subjected to 10,000 cycles of loading. Gross analysis demonstrated no implant extrusion, and further, that the hydrogel interdigitated well with native NP. Constructs were next surface-seeded with NP cells and cultured for 14 days, confirming lack of hydrogel cytotoxicity, with the hydrogel maintaining NP cell viability and promoting proliferation. Next, to evaluate the potential of the hydrogel to support cell-mediated matrix production, constructs were seeded with mesenchymal stem cells (MSCs) and cultured under prochondrogenic conditions for up to 42 days. Importantly, the hydrogel maintained MSC viability and promoted proliferation, as evidenced by increasing DNA content with culture duration. MSCs differentiated along a chondrogenic lineage, evidenced by upregulation of aggrecan and collagen II mRNA, and increased GAG and collagen content, and mechanical properties with increasing culture duration. Collectively, these results establish the therapeutic potential of this novel hydrogel for functional regeneration of the NP. Future work will confirm the ability of this hydrogel to normalize the mechanical stability of cadaveric human motion segments, and advance the material toward human translation using preclinical large-animal models.

Intervertebral disc degeneration has been implicated in the etiology of low back pain; however, the current surgical strategies for treating symptomatic disc disease are limited. A variety of materials have been developed to replace disc components, including the nucleus pulposus (NP), the annulus fibrosus (AF) and their combination into disc-like engineered constructs. We have previously shown that layers of electrospun poly(ε-caprolactone) scaffold, mimicking the hierarchical organization of the native AF, can achieve functional parity with native tissue. Likewise, we have combined these structures with cell-seeded hydrogels (as an NP replacement) to form disc-like angle-ply structures (DAPS). The objective of this study was to develop a model for the evaluation of DAPS in vivo. Through a series of studies, we developed a surgical approach to replace the rat caudal disc with an acellular DAPS and then stabilized the motion segment via external fixation. We then optimized cell infiltration into DAPS by including sacrificial poly(ethylene oxide) layers interspersed throughout the angle-ply structure. Our findings illustrate that DAPS are stable in the caudal spine, are infiltrated by cells from the peri-implant space and that infiltration is expedited by providing additional routes for cell migration. These findings establish a new in vivo platform in which to evaluate and optimize the design of functional disc replacements.

One of the most critical parameters in cartilage tissue engineering which influences the clinical success of a repair therapy is the ability to match the load-bearing capacity of the tissue as it functions in vivo. While mechanical forces are known to positively influence the development of cartilage matrix architecture, these same forces can induce long-term implant failure due to poor integration or structural deficiencies. As such, in the design of optimal repair strategies, it is critical to understand the timeline of construct maturation and how the elaboration of matrix correlates with the development of mechanical properties. We have previously characterized a scaffold-free method to engineer cartilage utilizing primary chondrocytes cultured at high density in hydrogel-coated culture vessels to promote the formation of a self-aggregating cell suspension that condenses to form a cartilage-like biomass, or cartilage tissue analog (CTA). Chondrocytes in these CTAs maintain their cellular phenotype and deposit extracellular matrix to form a construct that has characteristics similar to native cartilage; however, the mechanical integrity of CTAs had not yet been evaluated. In this study, we found that chondrocytes within CTAs produced a robust matrix of proteoglycans and collagen that correlated with increasing mechanical properties and decreasing cell-matrix ratios, leading to properties that approached that of native cartilage. These results demonstrate a unique approach to generating a cartilage-like tissue without the complicating factor of scaffold, while showing increased compressive properties and matrix characteristics consistent with other approaches, including scaffold-based constructs. To further improve the mechanics of CTAs, studies are currently underway to explore the effect of hydrodynamic loading and whether these changes would be reflective of in vivo maturation in animal models. The functional maturation of cartilage tissue analogs as described here support this engineered cartilage model for use in clinical and experimental applications for repair and regeneration in joint-related pathologies.

Given the limitations of current surgical approaches to treat articular cartilage injuries, tissue engineering (TE) approaches have been aggressively pursued. Despite reproduction of key mechanical attributes of native tissue, the ability of TE cartilage constructs to integrate with native tissue must also be optimized for clinical success. In this paper, we propose a "trajectory-based" tissue engineering (TB-TE) approach, based on the hypothesis that time-dependent increases in construct maturation in-vitro prior to implantation (i.e. positive rates) may provide a reliable predictor of in-vivo success. As an example TE system, we utilized hyaluronic acid hydrogels laden with mesenchymal stem cells. We first modeled the maturation of these constructs in-vitro to capture time-dependent changes. We then performed a sensitivity analysis of the model to optimize the timing and amount of data collection. Finally, we showed that integration to cartilage in-vitro is not correlated to the maturation state of TE constructs, but rather their maturation rate, providing a proof-of-concept for the use of TB-TE to enhance treatment outcomes following cartilage injury. This new approach challenges the traditional TE paradigm of matching only native state parameters of maturity and emphasizes the importance of also establishing an in-vitro trajectory in constructs in order to improve the chance of in-vivo success.

Fibrous scaffolds fabricated via electrospinning are being explored to repair injuries within dense connective tissues. However, there is still much to be understood regarding the appropriate scaffold properties that best support tissue repair. In this study, the influence of the stiffness of electrospun fibers on cell invasion into fibrous scaffolds is investigated. Specifically, soft and stiff electrospun fibrous networks are fabricated from crosslinked methacrylated hyaluronic acid (MeHA), where the stiffness is altered via the extent of MeHA crosslinking. Meniscal fibrochondrocyte (MFC) adhesion and migration into fibrous networks is investigated, where the softer MeHA fibrous networks are easily deformed and densified through cellular tractions and the stiffer MeHA fibrous networks support ~50% greater MFC invasion over weeks when placed adjacent to meniscal tissue. When the scaffolds are sandwiched between meniscal tissues and implanted subcutaneously, the stiffer MeHA fibrous networks again support enhanced cellular invasion and greater collagen deposition after 4 weeks when compared to the softer MeHA fibrous networks. These results indicate that the mechanics and deformability of fibrous networks likely alter cellular interactions and invasion, providing an important design parameter towards the engineering of scaffolds for tissue repair.

Harsh environments within damaged and diseased tissues and limited retention and survival of injected stem cells pose major challenges for stem cell therapeutics today. Here, we discuss promising hydrogel-based strategies for improving engraftment and viability of transplanted stem cells and stimulating recruitment of endogenous stem cells for repair.

Posttraumatic osteoarthritis (PTOA) occurs after traumatic injury to the joint. It is most common following injuries that disrupt the articular surface or lead to joint instability. The reported risk of PTOA following significant joint trauma is as high as 75%; articular fractures can increase the risk more than 20-fold. Despite recent advances in surgical management, the incidence of PTOA following intra-articular fractures has remained relatively unchanged over the last few decades. Pathogenesis of PTOA after intra-articular fracture is likely multifactorial and may be associated with acute cartilage injury as well as chronic joint overload secondary to instability, incongruity, and malalignment. Additional studies are needed to better elucidate how these factors contribute to the development of PTOA and to develop advanced treatment algorithms that consist of both acute biologic interventions targeted to decrease inflammation and cellular death in response to injury and improved surgical methods to restore stability, congruity, and alignment.

Mechanical deformation applied at the joint or tissue level is transmitted through the macroscale extracellular matrix to the microscale local matrix, where it is transduced to cells within these tissues and modulates tissue growth, maintenance, and repair. The objective of this study was to investigate how applied tissue strain is transferred through the local matrix to the cell and nucleus in meniscus, tendon, and the annulus fibrosus, as well as in stem cell-seeded scaffolds engineered to reproduce the organized microstructure of these native tissues. To carry out this study, we developed a custom confocal microscope-mounted tensile testing device and simultaneously monitored strain across multiple length scales. Results showed that mean strain was heterogeneous and significantly attenuated, but coordinated, at the local matrix level in native tissues (35–70% strain attenuation). Conversely, freshly seeded scaffolds exhibited very direct and uniform strain transfer from the tissue to the local matrix level (15–25% strain attenuation). In addition, strain transfer from local matrix to cells and nuclei was dependent on fiber orientation and tissue type. Histological analysis suggested that different domains exist within these fibrous tissues, with most of the tissue being fibrous, characterized by an aligned collagen structure and elongated cells, and other regions being proteoglycan (PG)-rich, characterized by a dense accumulation of PGs and rounder cells. In meniscus, the observed heterogeneity in strain transfer correlated strongly with cellular morphology, where rounder cells located in PG-rich microdomains were shielded from deformation, while elongated cells in fibrous microdomains deformed readily. Collectively, these findings suggest that different tissues utilize distinct strain-attenuating mechanisms according to their unique structure and cellular phenotype, and these differences likely alter the local biologic response of such tissues and constructs in response to mechanical perturbation.

Methacrylated hyaluronic acid (HA) hydrogels provide a backbone polymer with which mesenchymal stem cells (MSCs) can interact through several cell surface receptors that are expressed by MSCs, including CD44 and CD168. Previous studies showed that this 3D hydrogel environment supports the chondrogenesis of MSCs, and here we demonstrate through functional blockade that these specific cell–material interactions play a role in this process. Beyond matrix interactions, cadherin molecules, a family of transmembrane glycoproteins, play a critical role in tissue development during embryogenesis, and N-cadherin is a key factor in mediating cell–cell interactions during mesenchymal condensation and chondrogenesis. In this study, we functionalized HA hydrogels with N-cadherin mimetic peptides and evaluated their role in regulating chondrogenesis and cartilage matrix deposition by encapsulated MSCs. Our results show that conjugation of cadherin peptides onto HA hydrogels promotes both early chondrogenesis of MSCs and cartilage-specific matrix production with culture, compared with unmodified controls or those with inclusion of a scrambled peptide domain. This enhanced chondrogenesis was abolished via treatment with N-cadherin–specific antibodies, confirming the contribution of these N-cadherin peptides to chondrogenesis. Subcutaneous implantation of MSC-seeded constructs also showed superior neocartilage formation in implants functionalized with N-cadherin mimetic peptides compared with controls. This study demonstrates the inherent biologic activity of HA-based hydrogels, as well as the promise of biofunctionalizing HA hydrogels to emulate the complexity of the natural cell microenvironment during embryogenesis, particularly in stem cell-based cartilage regeneration.

Adult articular cartilage exhibits little capacity for intrinsic repair, and thus even minor injuries or lesions may lead to progressive damage and osteoarthritic joint degeneration, resulting in significant pain and disability. While there have been numerous attempts to develop tissue-engineered grafts or patches to repair focal chondral and osteochondral defects, there remain significant challenges in the clinical application of cell-based therapies for cartilage repair. This paper reviews the current state of cartilage tissue engineering with respect to different cell sources and their potential genetic modification, biomaterial scaffolds and growth factors, as well as preclinical testing in various animal models. This is not intended as a systematic review, rather an opinion of where the field is moving in light of current literature. While significant advances have been made in recent years, the complexity of this problem suggests that a multidisciplinary approach – combining a clinical perspective with expertise in cell biology, biomechanics, biomaterials science and high-throughput analysis will likely be necessary to address the challenge of developing functional cartilage replacements. With this approach we are more likely to realise the clinical goal of treating both focal defects and even large-scale osteoarthritic degenerative changes in the joint.

The field of tissue engineering and regenerative medicine (TERM) has exploded in the last decade. In this Year (or so) in Review, we highlight some of the high impact advances within the field over the past several years. Using the past as our guide and starting with an objective premise, we attempt so to identify recent “hot topics” and transformative publications within the field. Through this process, several key themes emerged: (1) tissue engineering: grafts and materials, (2) regenerative medicine: scaffolds and factors that control endogenous tissue formation, (3) clinical trials, and (4) novel cell sources: induced pluripotent stem cells. Within these focus areas, we summarize the highly impactful articles that emerged from our objective analysis and review additional recent publications to augment and expand upon these key themes. Finally, we discuss where the TERM field may be headed and how to monitor such a broad-based and ever-expanding community.

Biophysical stimuli presented to cells via microenvironmental properties (e.g., alignment and stiffness) or external forces have a significant impact on cell function and behavior. Recently, the cell nucleus has been identified as a mechanosensitive organelle that contributes to the perception and response to mechanical stimuli. However, the specific mechanotransduction mechanisms that mediate these effects have not been clearly established. Here, we offer a comprehensive review of the evidence supporting (and refuting) three hypothetical nuclear mechanotransduction mechanisms: physical reorganization of chromatin, signaling at the nuclear envelope, and altered cytoskeletal structure/tension due to nuclear remodeling. Our goal is to provide a reference detailing the progress that has been made and the areas that still require investigation regarding the role of nuclear mechanotransduction in cell biology. Additionally, we will briefly discuss the role that mathematical models of cell mechanics can play in testing these hypotheses and in elucidating how biophysical stimulation of the nucleus drives changes in cell behavior. While force-induced alterations in signaling pathways involving lamina-associated polypeptides (LAPs) (e.g., emerin and histone deacetylase 3 (HDAC3)) and transcription factors (TFs) located at the nuclear envelope currently appear to be the most clearly supported mechanism of nuclear mechanotransduction, additional work is required to examine this process in detail and to more fully test alternative mechanisms. The combination of sophisticated experimental techniques and advanced mathematical models is necessary to enhance our understanding of the role of the nucleus in the mechanotransduction processes driving numerous critical cell functions.

Dense fibrous connective tissues play critical load-bearing roles in the musculoskeletal system and represent a significant medical problem when their function is interrupted by progressive degeneration or acute injury. The intent of this chapter is to outline the functional aspects of two such tissues, the fibrocartilaginous knee meniscus and the annulus fibrosus of the intervertebral disc of the spine. In this chapter, we discuss the hallmark mechanical and biochemical features of each of these tissues, the incidence and most common means through which these tissues are damaged or degenerated, and the current clinical practices for their repair or replacement. Given the prevalence of damage to these tissues, numerous commercial products are, or will soon be, available in the marketplace; these products are discussed in the context of native tissue structure and function and the engineering principles and biomaterial components governing their design. Next, new products, based on tissue engineering strategies, are introduced, and their current status is explored with reference to clinical translation. Functional benchmarks, preclinical models, and evaluation tools, as well as future directions in this burgeoning field, are discussed.

The annulus fibrosus (AF) of the intervertebral disk plays a critical role in vertebral load transmission that is heavily dependent on the microscale structure and composition of the tissue. With degeneration, both structure and composition are compromised, resulting in a loss of AF mechanical function. Numerous tissue engineering strategies have addressed the issue of AF degeneration, but few have focused on recapitulation of AF microstructure and function. One approach that allows for generation of engineered AF with appropriate (+/-)30° lamellar microstructure is the use of aligned electrospun scaffolds seeded with mesenchymal stem cells (MSCs) and assembled into angle-ply laminates (APL). Previous work indicates that opposing lamellar orientation is necessary for development of near native uniaxial tensile properties. However, most native AF tensile loads are applied biaxially, as the disk is subjected to multi-axial loads and is constrained by its attachments to the vertebral bodies. Thus, the objective of this study was to evaluate the biaxial mechanical response of engineered AF bilayers, and to determine the importance of opposing lamellar structure under this loading regime. Opposing bilayers, which replicate native AF structure, showed a significantly higher modulus in both testing directions compared to parallel bilayers, and reached ∼60% of native AF biaxial properties. Associated with this increase in biaxial properties, significantly less shear, and significantly higher stretch in the fiber direction, was observed. These results provide additional insight into native tissue structure-function relationships, as well as new benchmarks for engineering functional AF tissue constructs.

Background This study compares a traditional parapatellar retinaculum-sacrificing arthrotomy to a retinaculum-sparing arthrotomy in a porcine stifle joint as a cartilage repair model. Findings Surgical exposure of the femoral trochlea of ten Yucatan pigs stifle joint was performed using either a traditional medial parapatellar approach with retinaculum incision and luxation of the patella (n = 5) or a minimally invasive (MIS) approach which spared the patellar retinaculum (n = 5). Both classical and MIS approaches provided adequate access to the trochlea, enabling the creation of cartilage defects without difficulties. Four full thickness, 4 mm circular full-thickness cartilage defects were created in each trochlea. There were no intraoperative complications observed in either surgical approach. All pigs were allowed full weight-bearing and full range of motion immediately postoperatively and were euthanized between 2 and 3 weeks. The traditional approach was associated with increased cartilage wear compared to the MIS approach. Two blinded raters performed gross evaluation of the trochlea cartilage surrounding the defects according to the modified ICRS cartilage injury classification. The traditional approach cartilage received a significantly worse score than the MIS approach group from both scorers (3.2 vs 0.8, p = 0.01 and 2.8 vs 0, p = 0.005 respectively). Conclusion The MIS approach results in less damage to the trochlear cartilage and faster return to load bearing activities. As an arthrotomy approach in the porcine model, MIS is superior to the traditional approach.

Extracellular matrix dynamics are key to tissue morphogenesis, homeostasis, injury, and repair. The spatiotemporal organization of this matrix has profound biological implications, but is challenging to monitor using standard techniques. Here, we address these challenges by using noncanonical amino acid tagging to fluorescently label extracellular matrix synthesized in the presence of bio-orthogonal methionine analogs. This strategy labels matrix proteins with high resolution, without compromising their distribution or mechanical function. We demonstrate that the organization and temporal dynamics of the proteinaceous matrix depend on the biophysical features of the microenvironment, including the biomaterial scaffold and the niche constructed by cells themselves. Pulse labeling experiments reveal that, in immature constructs, nascent matrix is highly fibrous and interdigitates with pre-existing matrix, while in more developed constructs, nascent matrix lacks fibrous organization and is retained in the immediate pericellular space. Inhibition of collagen crosslinking increases matrix synthesis, but compromises matrix organization. Finally, these data demonstrate marked cell-to-cell heterogeneity amongst both chondrocytes and mesenchymal stem cells undergoing chondrogenesis. Collectively, these results introduce fluorescent noncanonical amino acid tagging as a strategy to investigate spatiotemporal matrix organization, and demonstrate its ability to identify differences in phenotype, microenvironment, and matrix assembly at the single cell level.

Spinal conditions related to intervertebral disc (IVD) degeneration cost billions of dollars in the US annually. Despite the prevalence and soaring cost, there is no specific treatment that restores the physiological function of the diseased IVD. Thus, it is vital to develop new treatment strategies to repair the degenerating IVD. Persons with IVD degeneration without back pain or radicular leg pain often do not require any intervention. Only patients with severe back pain related to the IVD degeneration or biomechanical instability are likely candidates for cell therapy. The IVD progressively degenerates with age in humans, and strategies to repair the IVD depend on the stage of degeneration. Cell therapy and cell-based gene therapy aim to address moderate disc degeneration; advanced stage disease may require surgery. Studies involving autologous, allogeneic, and xenogeneic cells have all shown good survival of these cells in the IVD, confirming that the disc niche is an immunologically privileged site, permitting long-term survival of transplanted cells. All of the animal studies reviewed here reported some improvement in disc structure, and 2 studies showed attenuation of local inflammation. Among the 50 studies reviewed, 25 used some type of scaffold, and cell leakage is a consistently noted problem, though some studies showed reduced cell leakage. Hydrogel scaffolds may prevent cell leakage and provide biomechanical support until cells can become established matrix producers. However, these gels need to be optimized to prevent this leakage. Many animal models have been leveraged in this research space. Rabbit is the most frequently used model (28 of 50), followed by rat, pig, and dog. Sheep and goat IVDs resemble those of humans in size and in the absence of notochordal cells. Despite this advantage, there were only 2 sheep and 1 goat studies of 50 studies in this cohort. It is also unclear if a study in large animals is needed before clinical trials since some of the clinical trials proceeded without a study in large animals. No animal studies or clinical trials completely restored IVD structure. However, results suggest cause for optimism. In light of the fact that patients primarily seek medical care for back pain, attenuating local inflammation should be a priority in benchmarks for success. Clinicians generally agree that short-term back pain should be treated conservatively. When interventions are considered, the ideal therapy should also be minimally invasive and concurrent with other procedures such as discography or discectomy. Restoration of tissue structure and preservation of spinal motion are desirable.

Summary Objective The objective of this study was to establish a large animal model that recapitulates the spectrum of intervertebral disc degeneration that occurs in humans and which is suitable for pre-clinical evaluation of a wide range of experimental therapeutics. Design Degeneration was induced in the lumbar intervertebral discs of large frame goats by either intradiscal injection of chondroitinase ABC (ChABC) over a range of dosages (0.1U, 1U or 5U) or subtotal nucleotomy. Radiographs were used to assess disc height changes over 12 weeks. Degenerative changes to the discs and endplates were assessed via magnetic resonance imaging (MRI), semi-quantitative histological grading, microcomputed tomography (μCT), and measurement of disc biomechanical properties. Results Degenerative changes were observed for all interventions that ranged from mild (0.1U ChABC) to moderate (1U ChABC and nucleotomy) to severe (5U ChABC). All groups showed progressive reductions in disc height over 12 weeks. Histological scores were significantly increased in the 1U and 5U ChABC groups. Reductions in T2 and T1ρ, and increased Pfirrmann grade were observed on MRI. Resorption and remodeling of the cortical boney endplate adjacent to ChABC-injected discs also occurred. Spine segment range of motion (ROM) was greater and compressive modulus was lower in 1U ChABC and nucleotomy discs compared to intact. Conclusions A large animal model of disc degeneration was established that recapitulates the spectrum of structural, compositional and biomechanical features of human disc degeneration. This model may serve as a robust platform for evaluating the efficacy of therapeutics targeted towards varying degrees of disc degeneration.

Mesenchymal stem cell (MSC) differentiation is mediated by soluble and physical cues. In this study, we investigated differentiation-induced transformations in MSC cellular and nuclear biophysical properties and queried their role in mechanosensation. Our data show that nuclei in differentiated bovine and human MSCs stiffen and become resistant to deformation. This attenuated nuclear deformation was governed by restructuring of Lamin A/C and increased heterochromatin content. This change in nuclear stiffness sensitized MSCs to mechanical-loading-induced calcium signaling and differentiated marker expression. This sensitization was reversed when the ‘stiff’ differentiated nucleus was softened and was enhanced when the ‘soft’ undifferentiated nucleus was stiffened through pharmacologic treatment. Interestingly, dynamic loading of undifferentiated MSCs, in the absence of soluble differentiation factors, stiffened and condensed the nucleus, and increased mechanosensitivity more rapidly than soluble factors. These data suggest that the nucleus acts as a mechanostat to modulate cellular mechanosensation during differentiation. DOI: http://dx.doi.org/10.7554/eLife.18207.001

N-cadherin (Ncad) mediates cell-cell interactions, regulates β-catenin (βcat) signaling, and promotes the chondrogenic differentiation of mesenchymal lineage cells. Here, we utilized confocal imaging to investigate the influence of Ncad interactions on single mesenchymal stem cell (MSC) behavior within 3-dimensional hydrogel environments under conditions that promote chondrogenic differentiation. Human MSCs were photoencapsulated in hyaluronic acid (HA) hydrogels functionalized with Ncad mimetic peptides and compared to cells in environments with control non-active peptides (Ctrl). Using single-cell imaging, we observed a significant increase in membrane βcat, nuclear βcat, and cell roundness after 3 days in Ncad hydrogels compared to Ctrl hydrogels. The extent of membrane and nuclear βcat localization and MSC roundness decreased to Ctrl hydrogel levels via pre-treatment with Ncad-specific antibodies prior to encapsulation in the Ncad hydrogels, confirming the activity of the peptide. Interestingly, there was a pronounced (>80% increase) in βcat nuclear localization in two-cell clusters within the Ctrl hydrogels, which was much greater than the increase (~30%) in βcat nuclear localization in two-cell clusters within the Ncad hydrogels. In summary, we utilized fluorescent imaging to demonstrate Ncad-mediated single cell responses to developmental cues within hydrogels towards chondrogenesis.

Tissue fibrosis contributes to nearly half of all deaths in the developed world and is characterized by progressive matrix stiffening. Despite this, nearly all in vitro disease models are mechanically static. Here, we used visible light-mediated stiffening hydrogels to investigate cell mechanotransduction in a disease-relevant system. Primary hepatic stellate cell-seeded hydrogels stiffened in situ at later time points (following a recovery phase post-isolation) displayed accelerated signaling kinetics of both early (Yes-associated protein/Transcriptional coactivator with PDZ-binding motif, YAP/TAZ) and late (alpha-smooth muscle actin, α-SMA) markers of myofibroblast differentiation, resulting in a time course similar to observed in vivo activation dynamics. We further validated this system by showing that α-SMA inhibition following substrate stiffening resulted in attenuated stellate cell activation, with reduced YAP/TAZ nuclear shuttling and traction force generation. Together, these data suggest that stiffening hydrogels may be more faithful models for studying myofibroblast activation than static substrates and could inform the development of disease therapeutics.

In this work, a chemo-mechanical model describing the growth dynamics of cell-matrix adhesion structures (i.e. focal adhesions (FAs)) is developed. We show that there are three regimes for FA evolution depending on their size. Specifically, nascent adhesions with initial lengths below a critical value that are yet to engage in actin fibers will dissolve, whereas bigger ones will grow into mature FAs with a steady state size. In adhesions where growth surpasses the steady state size, disassembly will occur until their sizes are reduced back to the equilibrium state. This finding arises from the fact that polymerization of adhesion proteins is force-dependent. Under actomyosin contraction, individual integrin bonds within small FAs must transmit higher loads while the phenomenon of stress concentration occurs at the edge of large adhesion patches. As such, the effective stiffness of the FA-ECM complex that is either too small or too large will be relatively low, resulting in a limited actomyosin pulling force developed at the edge that is insufficient to prevent disassembly. Furthermore, it is found that a stiffer ECM and/or nucleus, as well as a stronger chemo-mechanical feedback, will induce larger adhesions along with a higher level of contraction force. Interestingly, switching the extracellular side from an elastic half-space, corresponding to some widely used in vitro gel substrates, to a 1D fiber does not qualitative change these conclusions. Our model predictions are in good agreement with a variety of experimental observations obtained in this study as well as those reported in the literature. Furthermore, this new model provides a framework in which to understand how both intracellular and extracellular perturbations lead to changes in adhesion structure number and size.

Hyaline cartilage serves as a low-friction and wear-resistant articulating surface in load-bearing, diarthrodial joints. Unfortunately, as the avascular, alymphatic nature of cartilage significantly impedes the body’s natural ability to regenerate, damage resulting from trauma and osteoarthritis necessitates repair attempts. Current clinical methods are generally limited in their ability to regenerate functional cartilage, and so research in recent years has focused on tissue engineering solutions in which the regeneration of cartilage is pursued through combinations of cells (e.g., chondrocytes or stem cells) paired with scaffolds (e.g., hydrogels, sponges, and meshes) in conjunction with stimulatory growth factors and bioreactors. A variety of synthetic and natural materials have been employed, most commonly in the form of hydrogels, and these systems have been tuned for optimal nutrient diffusion, connectivity of deposited matrix, degradation, soluble factor delivery, and mechanical loading for enhanced matrix production and organization. Even with these promising advances, the complex mechanical properties and biochemical composition of native cartilage have not been achieved, and engineering cartilage tissue still remains a significant challenge. Using hyaluronic acid hydrogels as an example, this review will follow the progress of material design specific to cartilage tissue engineering and propose possible future directions for the field.