Cynthia Gooch

Mechanical Stimulation of Tendon Tissue Engineered Constructs: Effects on Construct Stiffness, Repair Biomechanics, and Their Correlation

The objective of this study was to determine how in vitro mechanical stimulation of tissue engineered constructs affects their stiffness and modulus in culture and tendon repair biomechanics 12 weeks after surgical implantation. Using six female adult New Zealand White rabbits, autogenous tissue engineered constructs were created by seeding mesenchymal stem cells (0.1 x 10(6) cells/ml) in collagen gel (2.6 mg/ml) and combining both with a collagen sponge. Employing a novel experimental design strategy, four constructs from each animal were mechanically stimulated (one 1 Hz cycle every 5 min to 2.4% peak strain for 8 h/day for 2 weeks) while the other four remained unstretched during the 2 week culture period. At the end of incubation, three of the mechanically stimulated (S) and three of the nonstimulated (NS) constructs from each animal were assigned for in vitro mechanical testing while the other two autogenous constructs were implanted into bilateral full-thickness, full-length defects created in the central third of rabbit patellar tendons (PTs). No significant differences were found in the in vitro linear stiffnesses between the S (0.15+/-0.1 N/mm) and NS constructs (0.08+/-0.02 N/mm; mean+/-SD). However, in vitro mechanical stimulation significantly increased the structural and material properties of the repair tissue, including a 14% increase in maximum force (p=0.01), a 50% increase in linear stiffness (p=0.001), and 23-41% increases in maximum stress and modulus (p=0.01). The S repairs achieved 65%, 80%, 60%, and 40% of normal central PT maximum force, linear stiffness, maximum stress, and linear modulus, respectively. The results for the S constructs exceed values obtained previously by our group using the same animal and defect model, and to our knowledge, this is the first study to show the benefits of in vitro mechanical stimulation on tendon repair biomechanics. In addition, the linear stiffnesses for the construct and repair were positively correlated (r=0.56) as were their linear moduli (r=0.68). Such in vitro predictors of in vivo outcome hold the potential to speed the development of tissue engineered products by reducing the time and costs of in vivo studies.

Combined Effects of Scaffold Stiffening and Mechanical Preconditioning Cycles on Construct Biomechanics, Gene Expression, and Tendon Repair Biomechanics

Our group has previously reported that in vitro mechanical stimulation of tissue-engineered tendon constructs significantly increases both construct stiffness and the biomechanical properties of the repair tissue after surgery. When optimized using response surface methodology, our results indicate that a mechanical stimulus with three components (2.4% strain, 3000 cycles/day, and one cycle repetition) produced the highest in vitro linear stiffness. Such positive correlations between construct and repair stiffness after surgery suggest that enhancing structural stiffness before surgery could not only accelerate repair stiffness but also prevent premature failures in culture due to poor mechanical integrity. In this study, we examined the combined effects of scaffold crosslinking and subsequent mechanical stimulation on construct mechanics and biology. Autologous tissue-engineered constructs were created by seeding mesenchymal stem cells (MSCs) from 15 New Zealand white rabbits on type I collagen sponges that had undergone additional dehydrothermal crosslinking (termed ADHT in this manuscript). Both constructs from each rabbit were mechanically stimulated for 8h/day for 12 consecutive days with half receiving 100 cycles/day and the other half receiving 3000 cycles/day. These paired MSC-collagen autologous constructs were then implanted in bilateral full-thickness, full-length defects in the central third of rabbit patellar tendons. Increasing the number of in vitro cycles/day delivered to the ADHT constructs in culture produced no differences in stiffness or gene expression and no changes in biomechanical properties or histology 12 weeks after surgery. Compared to MSC-based repairs from a previous study that received no additional treatment in culture, ADHT crosslinking of the scaffolds actually lowered the 12-week repair stiffness. Thus, while ADHT crosslinking may initially stiffen a construct in culture, this specific treatment also appears to mask any benefits of stimulation among repairs postsurgery. Our findings emphasize the importance of properly preconditioning a scaffold to better control/modulate MSC differentiation in vitro and to further enhance repair outcome in vivo.

Mechanical Stimulation of Tissue Engineered Tendon Constructs: Effect of Scaffold Materials

Our group has shown that numerous factors can influence how tissue engineered tendon constructs respond to in vitro mechanical stimulation. Although one study showed that stimulating mesenchymal stem cell (MSC)-collagen sponge constructs significantly increased construct linear stiffness and repair biomechanics, a second study showed no such effect when a collagen gel replaced the sponge. While these results suggest that scaffold material impacts the response of MSCs to mechanical stimulation, a well-designed intra-animal study was needed to directly compare the effects of type-I collagen gel versus type-I collagen sponge in regulating MSC response to a mechanical stimulus. Eight constructs from each cell line (n=8 cell lines) were created in specially designed silicone dishes. Four constructs were created by seeding MSCs on a type-I bovine collagen sponge, and the other four were formed by seeding MSCs in a purified bovine collagen gel. In each dish, two cell-sponge and two cell-gel constructs from each line were then mechanically stimulated once every 5 min to a peak strain of 2.4%, for 8 h/day for 2 weeks. The other dish remained in an incubator without stimulation for 2 weeks. After 14 days, all constructs were failed to determine mechanical properties. Mechanical stimulation significantly improved the linear stiffness (0.048+/-0.009 versus 0.015+/-0.004; mean+/-SEM (standard error of the mean ) N/mm) and linear modulus (0.016+/-0.004 versus 0.005+/-0.001; mean+/-SEM MPa) of cell-sponge constructs. However, the same stimulus produced no such improvement in cell-gel construct properties. These results confirm that collagen sponge rather than collagen gel facilitates how cells respond to a mechanical stimulus and may be the scaffold of choice in mechanical stimulation studies to produce functional tissue engineered structures.

The relationships among spatiotemporal collagen gene expression, histology, and biomechanics following full-length injury in the murine patellar tendon

Tendon injuries are major orthopedic problems that worsen as the population ages. Type‐I (Col1) and type‐II (Col2) collagens play important roles in tendon midsubstance and tendon‐to‐bone insertion healing, respectively. Using double transgenic mice, this study aims to spatiotemporally monitor Col1 and Col2 gene expression, histology, and biomechanics up to 8 weeks following a full‐length patellar tendon injury. Gene expression and histology were analyzed weekly for up to 5 weeks while mechanical properties were measured at 1, 2, 5, and 8 weeks. At week 1, the healing region displayed loose granulation tissue with little Col1 expression. Col1 expression peaked at 2 weeks, but the ECM was highly disorganized and hypercellular. By 3 weeks, Col1 expression had reduced and by 5 weeks, the ECM was generally aligned along the tendon axis. Col2 expression was not seen in the healing midsubstance or insertion at any time point. The biomechanics of the healing tissue was inadequate at all time points, achieving ultimate loads and stiffnesses of 48% and 63% of normal values by 8 weeks. Future studies will further characterize the cells within the healing midsubstance and insertion using tenogenic markers and compare these results to those of tendon cells during normal development.

Improving linear stiffness of the cell-seeded collagen sponge constructs by varying the components of the mechanical stimulus.

In vitro mechanical stimulation has been reported to induce cell alignment and increase cellular proliferation and collagen synthesis. Our group has previously reported that in vitro mechanical stimulation of tissue-engineered tendon constructs significantly increases construct stiffness and repair biomechanics after surgery. However, these studies used a single mechanical stimulation profile, the latter composed of multiple components whose individual and combined effects on construct properties remain unknown. Thus, the purpose of this study was to understand the relative importance of a subset of these components on construct stiffness. To try to optimize the resulting mechanical stimulus, we used an iterative process to vary peak strain, cycle number, and cycle repetition while controlling cycle frequency (1 Hz), rise and fall times (25% and 17% of the period, respectively), hours of stimulation/day (8 h/day), and total time of stimulation (12 days). Two levels of peak strain (1.2 % and 2.4%), cycle number (100 and 3000 cycles/day), and cycle repetition (1 and 20) were first examined. Higher levels of peak strain and cycle number were then examined to optimize the stimulus using response surface methodology. Our results indicate that constructs stimulated with 2.4% strain, 3000 cycles/day, and one cycle repetition produced the stiffest constructs. Given the significant positive correlations we have previously found between construct stiffness and repair biomechanics at 12 weeks post-surgery, these in vitro enhancements offer the prospect of further improving repair biomechanics.

Functional tissue engineering of tendon: Establishing biological success criteria for improving tendon repair.

Improving tendon repair using Functional Tissue Engineering (FTE) principles has been the focus of our laboratory over the last decade. Although our primary goals were initially focused only on mechanical outcomes, we are now carefully assessing the biological properties of our tissue-engineered tendon repairs so as to link biological influences with mechanics. However, given the complexities of tendon development and healing, it remains challenging to determine which aspects of tendon biology are the most important to focus on in the context of tissue engineering. To address this problem, we have formalized a strategy to identify, prioritize, and evaluate potential biological success criteria for tendon repair. We have defined numerous biological properties of normal tendon relative to cellular phenotype, extracellular matrix and tissue ultra-structure that we would like to reproduce in our tissue-engineered repairs and prioritized these biological criteria by examining their relative importance during both normal development and natural tendon healing. Here, we propose three specific biological criteria which we believe are essential for normal tendon function: (1) scleraxis-expressing cells; (2) well-organized and axially-aligned collagen fibrils having bimodal diameter distribution; and (3) a specialized tendon-to-bone insertion site. Moving forward, these biological success criteria will be used in conjunction with our already established mechanical success criteria to evaluate the effectiveness of our tissue-engineered tendon repairs.

Improved biomechanical and biological outcomes in the MRL/MpJ murine strain following a full‐length patellar tendon injury

Musculoskeletal injuries greatly affect the U.S. population and current clinical approaches fail to restore long‐term native tissue structure and function. Tissue engineering is a strategy advocated to improve tendon healing; however, the field still needs to establish biological benchmarks for assessing the effectiveness of tissue‐engineered structures. Investigating superior healing models, such as the MRL/MpJ, offers the opportunity to first characterize successful healing and then apply experimental findings to tissue‐engineered therapies. This study seeks to evaluate the MRL/MpJs healing response following a central patellar tendon injury compared to wildtype. Gene expression and histology were assessed at 3, 7, and 14 days following injury and mechanical properties were measured at 2, 5, and 8 weeks. Native patellar tendon biological and mechanical properties were not different between strains. Following injury, the MRL/MpJ displayed increased mechanical properties between 5 and 8 weeks; however, early tenogenic expression patterns were not different between the strains. Furthermore, expression of the cyclin‐dependent kinase inhibitor, p21, was not different between strains, suggesting an alternative mechanism may be driving the healing response. Future studies will investigate collagen structure and alignment of the repair tissue and characterize the complete healing transcriptome to identify mechanisms driving the MRL/MpJ response.

Evolving Strategies in Mechanobiology to More Effectively Treat Damaged Musculoskeletal Tissues

In this paper, we had four primary objectives. (1) We reviewed a brief history of the Lissner award and the individual for whom it is named, H.R. Lissner. We examined the type (musculoskeletal, cardiovascular, and other) and scale (organism to molecular) of research performed by prior Lissner awardees using a hierarchical paradigm adopted at the 2007 Biomechanics Summit of the US National Committee on Biomechanics. (2) We compared the research conducted by the Lissner award winners working in the musculoskeletal (MS) field with the evolution of our MS research and showed similar trends in scale over the past 35 years. (3) We discussed our evolving mechanobiology strategies for treating musculoskeletal injuries by accounting for clinical, biomechanical, and biological considerations. These strategies included studies to determine the function of the anterior cruciate ligament and its graft replacements as well as novel methods to enhance soft tissue healing using tissue engineering, functional tissue engineering, and, more recently, fundamental tissue engineering approaches. (4) We concluded with thoughts about future directions, suggesting grand challenges still facing bioengineers as well as the immense opportunities for young investigators working in musculoskeletal research. Hopefully, these retrospective and prospective analyses will be useful as the ASME Bioengineering Division charts future research directions.

The LG/J murine strain exhibits near-normal tendon biomechanical properties following a full-length central patellar tendon defect.

ABSTRACT Purpose of the study: Identifying biological success criteria is needed to improve therapies, and one strategy for identifying them is to analyze the RNA transcriptome for successful and unsuccessful models of tendon healing. We have characterized the MRL/MpJ murine strain and found improved mechanical outcomes following a central patellar tendon (PT) injury. In this study, we evaluate the healing of the LG/J murine strain, which comprises 75% of the MRL/MpJ background, to determine if the LG/J also exhibits improved biomechanical properties following injury and to determine differentially expressed transcription factors across the C57BL/6, MRL/MpJ and the LG/J strains during the early stages of healing. Materials and Methods: A full-length, central PT defect was created in 16–20 week old MRL/MpJ, LG/J, and C57BL/6 murine strains. Mechanical properties were assessed at 2, 5, and 8 weeks post surgery. Transcriptomic expression was assessed at 3, 7, and 14 days following injury using a novel clustering software program to evaluate differential expression of transcription factors. Results: Average LG/J structural properties improved to 96.7% and 97.2% of native LG/J PT stiffness and ultimate load by 8 weeks post surgery, respectively. We found the LG/J responded by increasing expression of transcription factors implicated in the inflammatory response and collagen fibril organization. Conclusions: The LG/J strain returns to normal structural properties by 8 weeks, with steadily increasing properties at each time point. Future work will characterize the cell populations responding to injury and investigate the role of the differentially expressed transcription factors during healing.

The Relationships Among Spatiotemporal Gene Expression, Histology, and Biomechanics Following Full-Length Injury in the Murine Patellar Tendon

Tendon and ligament injuries present a considerable socioeconomic impact as close to 50% of the 32 million musculoskeletal injuries in the US per year include these structures [1]. The inadequate healing in these tissues requires novel treatment modalities. Improving tendon tissue engineering dictates that we better understand the process of natural adult tendon healing. Type-I (Col1) and Type-II (Col2) collagens are important structural proteins in tendon as Col1 is the main collagen type found in the tendon midsubstance while Col2 is expressed at the insertion into bone during development, growth, and healing [2–3]. Expression of Col1 and Col2 has typically been analyzed via qPCR, western blotting, and immunohistochemistry (IHC) during healing. However, the temporal expression of these genes is still poorly understood on a cell-by-cell basis. Our lab has previously studied patellar tendon (PT) healing in NZW rabbits [4]. While the NZW rabbit allows for controlled injuries and accurate biomechanical assessment of healing, it lacks the genetic power that is offered in the mouse. Therefore, pOBCo13.6GFPtpz (Col1) and pCol2ECFP (Col2) double transgenic (DT) reporter mice were created to track spatiotemporal gene expression. Thus, the objectives of this study were to monitor changes in: 1) spatiotemporal Col1 and Col2 gene expression patterns, 2) tissue morphology, and 3) healing biomechanics following a full-length, central PT injury in Col1/Col2 DT mice and to compare these natural healing results to contralateral surgical shams and normal PT in age-matched controls.Copyright