Natalia Juncosa-Melvin

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.

Effect of scaffold material, construct length and mechanical stimulation on the in vitro stiffness of the engineered tendon construct

Introducing mesenchymal stem cell (MSC)-seeded collagen constructs into load-protected wound sites in the rabbit patellar and Achilles tendons significantly improves their repair outcome compared to natural healing of the unfilled defect. However, these constructs would not be acceptable alternatives for repairing complete tendon ruptures because they lack the initial stiffness at the time of surgery to resist the expected peak in vivo forces thereafter. Since the stiffness of these constructs has also been shown to positively correlate with the stiffness of the subsequent repairs, improving initial stiffness by appropriate selection of in vitro culture conditions would seem crucial. In this study we examined the individual and combined effects of collagen scaffold type, construct length, and mechanical stimulation on in vitro implant stiffness. Two levels each of scaffold material (collagen gel vs. collagen sponge), construct length (short vs. long), and mechanical stimulation (stimulated vs. non-stimulated) were examined. Our results indicate that all three treatment factors influenced construct linear stiffness. Increasing the length of the construct had the greatest effect on the stiffness compared to introducing mechanical stimulation or changing the scaffold material. A significant interaction was also found between length and stimulation. Of the eight groups studied, longer, stimulated, cell-sponge constructs showed the highest in vitro linear stiffness. We now plan in vivo studies to determine if higher stiffness constructs generate higher stiffness repairs 12 weeks after surgery and if in vitro construct stiffness continues to correlate with in vivo repair parameters like linear stiffness.

Using Functional Tissue Engineering and Bioreactors to Mechanically Stimulate Tissue-Engineered Constructs

Bioreactors precondition tissue-engineered constructs (TECs) to improve integrity and hopefully repair. In this paper, we use functional tissue engineering to suggest criteria for preconditioning TECs. Bioreactors should (1) control environment during mechanical stimulation; (2) stimulate multiple constructs with identical or individual waveforms; (3) deliver precise displacements, including those that mimic in vivo activities of daily living (ADLs); and (4) adjust displacement patterns based on reaction loads and biological activity. We apply these criteria to three bioreactors. We have placed a pneumatic stimulator in a conventional incubator and stretched four constructs in each of five silicone dishes. We have also programmed displacement-limited stimuli that replicate frequencies and peak in vivo patellar tendon (PT) strains. Cellular activity can be monitored from spent media. However, our design prevents direct TEC force measurement. We have improved TEC stiffness as well as PT repair stiffness and shown correlations between the two. We have also designed an incubator to fit within each of two electromagnetic stimulators. Each incubator provides cell viability like a commercial incubator. Multiple constructs are stimulated with precise displacements that can mimic ADL strain patterns and record individual forces. Future bioreactors could be further improved by controlling and measuring TEC displacements and forces to create more functional tissues for surgeons and their patients.

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.

Chondroitin-6-Sulfate Incorporation and Mechanical Stimulation Increase MSC-Collagen Sponge Construct Stiffness

Using functional tissue engineering principles, our laboratory has produced tendon repair tissue which matches the normal patellar tendon force‐displacement curve up to 32% of failure. This repair tissue will need to withstand more strenuous activities, which can reach or even exceed 40% of failure force. To improve the linear stiffness of our tissue engineered constructs (TECs) and tissue engineered repairs, our lab is incorporating the glycosaminoglycan chondroitin‐6‐sulfate (C6S) into a type I collagen scaffold. In this study, we examined the effect of C6S incorporation and mechanical stimulation cycle number on linear stiffness and mRNA expression (collagen types I and III, decorin and fibronectin) for mesenchymal stem cell (MSC)‐collagen sponge TECs. The TECs were fabricated by inoculating MSCs at a density of 0.14 × 106 cells/construct onto pre‐cut scaffolds. Primarily type I collagen scaffold materials, with or without C6S, were cultured using mechanical stimulation with three different cycle numbers (0, 100, or 3,000 cycles/day). After 2 weeks in culture, TECs were evaluated for linear stiffness and mRNA expression. C6S incorporation and cycle number each played an important role in gene expression, but only the interaction of C6S incorporation and cycle number produced a benefit for TEC linear stiffness.

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.

An artificial tendon with durable muscle interface

A coupling mechanism that can permanently fix a forcefully contracting muscle to a bone anchor or any totally inert prosthesis would meet a serious need in orthopaedics. Our group developed the OrthoCoupler™ device to satisfy these demands. The objective of this study was to test OrthoCouplers performance in vitro and in vivo in the goat semitendinosus tendon model. For in vitro evaluation, 40 samples were fatigue‐tested, cycling at 10 load levels, n = 4 each. For in vivo evaluation, the semitendinosus tendon was removed bilaterally in eight goats. Left sides were reattached with an OrthoCoupler, and right sides were reattached using the Krackow stitch with #5 braided polyester sutures. Specimens were harvested 60 days postsurgery and assigned for biomechanics and histology. Fatigue strength of the devices in vitro was several times the contractile force of the semitendinosus muscle. The in vivo devices were built equivalent to two of the in vitro devices, providing an additional safety factor. In strength testing at necropsy, suture controls pulled out at 120.5 ± 68.3 N, whereas each OrthoCoupler was still holding after the muscle tore, remotely, at 298 ± 111.3 N (mean ± SD) (p < 0.0003). Muscle tear strength was reached with the fiber–muscle composite produced in healing still soundly intact. This technology may be of value for orthopaedic challenges in oncology, revision arthroplasty, tendon transfer, and sports‐injury reconstruction.

An Artificial Tendon to Connect the Quadriceps Muscle to the Tibia

No permanent, reliable artificial tendon exists clinically. Our group developed the OrthoCoupler™ device as a versatile connector, fixed at one end to a muscle, and adaptable at the other end to inert implants such as prosthetic bones or to bone anchors. The objective of this study was to evaluate four configurations of the device to replace the extensor mechanism of the knee in goats. Within muscle, the four groups had: (A) needle‐drawn uncoated bundles, (B) needle‐drawn coated bundles, (C) barbed uncoated bundles, and (D) barbed coated bundles. The quadriceps tendon, patella, and patellar tendon were removed from the right hind limb in 24 goats. The four groups (n = 6 for each) were randomly assigned to connect the quadriceps muscle to the tibia (with a bone plate). Specimens were collected from each operated leg and contralateral unoperated controls both for mechanical testing and histology at 90 days post‐surgery. In strength testing, maximum forces in the operated leg (vs. unoperated control) were 1,288 ± 123 N (vs. 1,387 ± 118 N) for group A, 1,323 ± 144 N (vs. 1,396 ± 779 N) for group B, 930 ± 125 N (vs. 1,337 ± 126 N) for group C, and 968 ± 109 N (vs. 1,528 ± 146 N) for group D (mean ± SEM). The strengths of the OrthoCoupler™ legs in the needled device groups were equivalent to unoperated controls (p = 0.6), while both barbed device groups had maximum forces significantly lower than their controls (p = 0.001). We believe this technology will yield improved procedures for clinical challenges in orthopaedic oncology, revision arthroplasty, tendon transfer, and tendon injury reconstruction. © 2011 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 29:1775–1782, 2011

A soft-tissue coupling for wound closure

Wounds often cannot be successfully closed by conventional means of closure such as sutures or staples. Our group developed the FiberSecure™ device to close soft tissue wounds reliably, surpassing native tissue strength. We closed cross-fiber muscle incisions, to evaluate (1) four different configurations of FiberSecure™ for 30 days, then (2) the resulting preferred configuration for 180 days. The four treatment groups each placed 21,504 polyester (PET) 12-μm fibers (cross-sectional area 1% of muscle) traversing the incision, in the form of (A) Four large (No.7 suture) non-textured bundles, (B) Eight small (No.2 suture) non-textured, (C) Four large textured, or (D) Eight small textured. Four incisions were closed in the external oblique muscle of 16 Sinclair minipigs. At 30 days, specimens were removed for biomechanics, histology, and total collagen content. Group (B) was selected for 180-day evaluations in the same wound model in eight animals, four closures each (n = 32), again with biomechanics and histology. In strength testing, every specimen tore through muscle remotely, while the repair region remained intact. Maximum forces were (A) 37.8 ± 3.9 N, (B) 37.1 ± 4.7 N, (C) 39.0 ± 5.3 N, and (D) 32.4 ± 3.4 N at 30 days, and 37.2 ± 11.3 N at 180 days (mean ± SEM). No significant difference was observed among the groups or time points (p > 0.05).