RESUMEN
Reliability of wound closure is limited primarily by the capacity of tissues to support conventional sutures (or staples), not by strength of either material per se. We developed FiberSecure™ for closures to surpass tissue strength. We assessed and compared the mechanical and histological performance of FiberSecure™ suture versus commercially available braided polyester suture (Mersilene) in the closure of abdominal muscle incisions in miniature swine at approximately 3 months postsurgery. Four incisions were closed in the external oblique muscle of eight Sinclair minipigs. Two wounds were closed with FiberSecure™ suture size 0 and the remaining two with Mersilene suture size 0. At 90 days, specimens were removed for biomechanics and histology. In destructive tensile testing, in the 16 abdominal muscle specimens for the FiberSecure™ suture, muscle tear was not near the suture implantation region, which remained intact. Wound strength met or exceeded strength of neighboring tissue in FiberSecure™ groups, which had peak force of 55.7 ± 22.1 N (mean ± SD) and peak stress of 579.0 ± 159.2 KPa (mean ± SD). For Mersilene, 3 of the 16 samples tore at the suture site and the remaining samples tore through the abdominal muscle not near the implantation region. The wound strength was similar to surrounding tissue, and these specimens had peak force of 51.8 ± 21.7 N and peak stress of 550.3 ± 239.4 KPa (mean ± SD). No significant difference was observed in peak force or stress between groups (p > 0.05), most repairs having met or exceeded native tissue strength by this time point. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 1126-1130, 2017.
Asunto(s)
Ensayo de Materiales , Poliésteres , Suturas , Técnicas de Cierre de Heridas , Animales , Porcinos , Porcinos EnanosRESUMEN
Whenever a tendon or its bone insertion is disrupted or removed, existing surgical techniques provide a temporary connection or scaffolding to promote healing, but the interface of living to non-living materials soon breaks down under the stress of these applications, if it must bear the load more than acutely. Patients are thus disabled whose prostheses, defect size, or mere anatomy limit the availability or outcomes of such treatments. Our group developed the OrthoCoupler™ device to join skeletal muscle to prosthetic or natural structures without this interface breakdown. In this study, the goat knee extensor mechanism (quadriceps tendon, patella, and patellar tendon) was removed from the right hind limb in 16 goats. The device connected the quadriceps muscle to a stainless steel bone plate on the tibia. Mechanical testing and histology specimens were collected from each operated leg and contralateral unoperated control legs at 180 days. Maximum forces in the operated leg (vs. unoperated) were 1,400 ± 93 N (vs. 1,179 ± 61 N), linear stiffnesses were 33 ± 3 N/mm (vs. 37 ± 4 N/mm), and elongations at failure were 92.1 ± 5.3 mm (vs. 68.4 ± 3.8 mm; mean ± SEM). Higher maximum forces (p = 0.02) and elongations at failure (p=0.008) of legs with the device versus unoperated controls were significant; linear stiffnesses were not (p=0.3). We believe this technology will yield improved procedures for clinical challenges in orthopedic oncology, revision arthroplasty, tendon transfer, and tendon injury reconstruction.
Asunto(s)
Órganos Artificiales/normas , Procedimientos Ortopédicos/métodos , Músculo Cuádriceps/cirugía , Tendones/cirugía , Tibia/cirugía , Cicatrización de Heridas/fisiología , Animales , Fenómenos Biomecánicos/fisiología , Cabras , Recuperación del Miembro/métodos , Masculino , Ensayo de Materiales , Implantación de Prótesis/métodos , Implantación de Prótesis/normas , Reproducibilidad de los Resultados , Tendones/fisiología , Soporte de Peso/fisiologíaRESUMEN
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.
Asunto(s)
Órganos Artificiales , Recuperación del Miembro/métodos , Diseño de Prótesis , Músculo Cuádriceps/cirugía , Tendones , Tibia/cirugía , Animales , Fenómenos Biomecánicos/fisiología , Materiales Biocompatibles Revestidos , Análisis de Falla de Equipo , Cabras , Recuperación del Miembro/instrumentación , Masculino , Modelos Animales , Actividad Motora/fisiología , Procedimientos Ortopédicos/instrumentación , Procedimientos Ortopédicos/métodos , Oseointegración , Músculo Cuádriceps/fisiología , Estrés Mecánico , Tibia/fisiologíaRESUMEN
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).
Asunto(s)
Vendajes , Ensayo de Materiales/métodos , Músculo Esquelético/lesiones , Heridas Penetrantes/terapia , Animales , Femenino , Músculo Esquelético/patología , Porcinos , Porcinos Enanos , Factores de Tiempo , Heridas Penetrantes/patologíaRESUMEN
Tendon and ligament injuries are significant contributors to musculoskeletal injuries. Unfortunately, traditional methods of repair are not uniformly successful and can require revision surgery. Our research is focused on identifying appropriate animal injury models and using tissue-engineered constructs (TECs) from bone-marrow-derived mesenchymal stem cells and collagen scaffolds. Critical to this effort has been the development of functional tissue engineering (FTE). We first determine the in vivo mechanical environment acting on the tissue and then precondition the TECs in culture with aspects of these mechanical signals to improve repair outcome significantly. We describe here a detailed protocol for conducting several complete iterations around our FTE 'road map.' The in vitro portion, from bone marrow harvest to TEC collection, takes 54 d. The in vivo portion, from TEC implantation to limb harvest, takes 84 d. One complete loop around the tissue engineering road map, as presented here, takes 138 d to complete.
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Colágeno/fisiología , Trasplante de Células Madre Mesenquimatosas/métodos , Células Madre Mesenquimatosas/fisiología , Traumatismos de los Tendones/terapia , Ingeniería de Tejidos/métodos , Animales , Materiales Biocompatibles , Fenómenos Biomecánicos , Bioprótesis , Femenino , Implantes Experimentales , Células Madre Mesenquimatosas/citología , Conejos , Andamios del TejidoRESUMEN
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 x 10(6) 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.
Asunto(s)
Sulfatos de Condroitina/metabolismo , Colágeno/fisiología , Andamios del Tejido , Animales , Fenómenos Biomecánicos , Sulfatos de Condroitina/farmacología , Colágeno Tipo I/biosíntesis , Colágeno Tipo III/biosíntesis , Decorina , Proteínas de la Matriz Extracelular/biosíntesis , Femenino , Fibronectinas/biosíntesis , Células Madre Mesenquimatosas/fisiología , Proteoglicanos/biosíntesis , Conejos , Resistencia a la Tracción , Ingeniería de Tejidos , Andamios del Tejido/químicaRESUMEN
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 OrthoCoupler's 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.
Asunto(s)
Ensayo de Materiales , Procedimientos Ortopédicos/instrumentación , Procedimientos de Cirugía Plástica/instrumentación , Diseño de Prótesis , Implantación de Prótesis/instrumentación , Traumatismos de los Tendones/cirugía , Animales , Materiales Biocompatibles , Fenómenos Biomecánicos , Modelos Animales de Enfermedad , Análisis de Falla de Equipo , Músculo Esquelético/patología , Músculo Esquelético/cirugía , Implantación de Prótesis/métodos , Procedimientos de Cirugía Plástica/métodos , Estrés Mecánico , Traumatismos de los Tendones/patología , Tendones/patología , Tendones/cirugíaRESUMEN
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.
Asunto(s)
Regulación de la Expresión Génica , Tendones/patología , Andamios del Tejido , Cicatrización de Heridas , Animales , Fenómenos Biomecánicos , Inmunohistoquímica , Conejos , Ingeniería de TejidosRESUMEN
The objectives of this study were to determine how tensile stimulation delivered up to 14 days in culture influenced type I collagen gene expression in stem cells cultured in collagen sponges, and to establish if gene expression, measured using a fluorescence method, correlates with an established method, real-time quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). Using a novel model system, mesenchymal stem cells were harvested from six double transgenic mice in which the type I and type II collagen promoters were linked to green fluorescent protein-topaz and enhanced cyan fluorescent protein, respectively. Tissue-engineered constructs were created by seeding 0.5 x 10(6) mesenchymal stem cells onto type I collagen sponge scaffolds in a silicone dish. Constructs were then transferred to a custom pneumatic mechanical stimulation system housed in a standard incubator and stimulated for 5 h=day in tension for either 7 or 14 days using a repeated profile (2.4% peak strain for 20 s at 1 Hz followed by a rest period at 0% strain for 100 s). Control specimens were exposed to identical culture conditions but without mechanical stimulation. At three time points (0, 7, and 14 days), constructs were then prepared for evaluation of gene expression using fluorescence analysis and qRT-PCR, and the remaining constructs were failed in tension. Both analytical methods showed that constructs stimulated for 7 and 14 days showed significantly higher collagen type I gene expression than nonstimulated controls at the same time interval. Gene expression measured using qRT-PCR and fluorescence analysis was positively correlated (r = 0.9). Linear stiffness of stimulated constructs was significantly higher at both 7 and 14 days than that of nonstimulated controls at the same time intervals. Linear stiffness of the stimulated constructs at day 14 was significantly different from that of day 7. Future studies will vary the mechanical signal to optimize type I collagen gene expression to improve construct biomechanics and in vivo tendon repair.
Asunto(s)
Colágeno Tipo I/genética , Regulación de la Expresión Génica , Poríferos/química , Células Madre/citología , Células Madre/metabolismo , Resistencia a la Tracción , Andamios del Tejido/química , Animales , Colágeno Tipo I/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Ratones , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
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.
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Reactores Biológicos , Ingeniería de Tejidos/instrumentación , Ingeniería de Tejidos/métodos , Andamios del Tejido , Animales , Células Cultivadas , Femenino , ConejosRESUMEN
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.
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Colágeno/química , Células Madre Mesenquimatosas/citología , Estrés Mecánico , Ingeniería de Tejidos/métodos , Animales , Fenómenos Biomecánicos , Células Cultivadas , Femenino , Células Madre Mesenquimatosas/fisiología , ConejosRESUMEN
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.
Asunto(s)
Trasplante de Células Madre Mesenquimatosas/métodos , Tendones/fisiología , Ingeniería de Tejidos , Andamios del Tejido , Animales , Fenómenos Biomecánicos , Línea Celular , Células Cultivadas , Colágeno Tipo I/fisiología , Elasticidad , Femenino , Geles , Rótula/lesiones , Rótula/fisiología , Conejos , Traumatismos de los Tendones/fisiopatología , Traumatismos de los Tendones/cirugíaRESUMEN
Over the past 8 years, our group has been continuously improving tendon repair using a functional tissue engineering (FTE) paradigm. This paradigm was motivated by inconsistent clinical results after tendon repair and reconstruction, and the modest biomechanical improvements we observed after repair of rabbit central patellar tendon defects using mesenchymal stem cell-gel-suture constructs. Although possessing a significantly higher stiffness and failure force than for natural healing, these first generation constructs were quite weak compared to normal tendon. Fundamental to the new FTE paradigm was the need to determine in vivo forces to which the repair tissue might be exposed. We first recorded these force patterns in two normal tendon models and then compared these peak forces to those for repairs of central defects in the rabbit patellar tendon model (PT). Replacing the suture with end-posts in culture and lowering the mesenchymal stem cell (MSC) concentration of these constructs resulted in failure forces greater than peak in vivo forces that were measured for all the studied activities. Augmenting the gel with a type I collagen sponge further increased repair stiffness and maximum force, and resulted in the repair tangent stiffness matching normal stiffness up to peak in vivo forces. Mechanically stimulating these constructs in bioreactors further enhanced repair biomechanics compared to normal. We are now optimizing components of the mechanical signal that is delivered in culture to further improve construct and repair outcome. Our contributions in the area of tendon functional tissue engineering have the potential to create functional load-bearing repairs that will revolutionize surgical reconstruction after tendon and ligament injury.
Asunto(s)
Trasplante de Células Madre Mesenquimatosas , Traumatismos de los Tendones/cirugía , Traumatismos de los Tendones/terapia , Ingeniería de Tejidos/métodos , Andamios del Tejido , Animales , Distinciones y Premios , Fenómenos Biomecánicos , Reactores Biológicos , Terapia Combinada , Estimulación Física/métodos , Estrés Mecánico , Traumatismos de los Tendones/fisiopatologíaRESUMEN
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.
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Bioprótesis , Traumatismos de los Tendones/rehabilitación , Resistencia a la Tracción , Ingeniería de Tejidos/métodos , Cicatrización de Heridas , Animales , Materiales Biocompatibles/metabolismo , Materiales Biocompatibles/uso terapéutico , Técnicas de Cultivo de Célula , Modelos Animales de Enfermedad , Elasticidad , Femenino , Implantes Experimentales , Ensayo de Materiales , Trasplante de Células Madre Mesenquimatosas , Células Madre Mesenquimatosas , Ligamento Rotuliano/lesiones , Ligamento Rotuliano/cirugía , Conejos , Traumatismos de los Tendones/cirugía , Andamios del TejidoRESUMEN
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.
Asunto(s)
Tendones , Resistencia a la Tracción/fisiología , Ingeniería de Tejidos/métodos , Andamios del Tejido , Animales , Materiales Biocompatibles/química , Materiales Biocompatibles/metabolismo , Células Cultivadas , Colágeno Tipo I/química , Elasticidad , Femenino , Geles/química , Trasplante de Células Madre Mesenquimatosas , Células Madre Mesenquimatosas/química , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/metabolismo , Conejos , Estrés Mecánico , Tendones/química , Tendones/citología , Tendones/metabolismo , Ingeniería de Tejidos/instrumentación , TransductoresRESUMEN
Our group has shown that mechanical stimulation increases the stiffness of stem cell-collagen sponge constructs at 14 days in culture and subsequent rabbit patellar tendon repairs at 12 weeks postsurgery. What remains unclear is which genes might be responsible for this increase in stiffness. Therefore, the objective of this study was to determine how a tensile stimulus affects the gene expression of stem cell-collagen sponge constructs used to repair rabbit central patellar tendon defects. Tissue-engineered constructs were created by seeding mesenchymal stem cells (MSCs) from 10 adult rabbits at 0.14 x 10(6) cells/construct in type I collagen sponges. Half of the constructs were mechanically stimulated once every 5 min for 8 h/d to a peak strain of 2.4% for 2 weeks. The other half remained in an incubator without mechanical stimulation for 2 weeks. After 14 days in culture, half of the stimulated and nonstimulated constructs were prepared to determine the expression of collagen type I, collagen type III, decorin, fibronectin, and glyceraldehyde-3-phosphate dehydrogenase genes using real-time quantitative reverse transcriptase polymerase chain reaction. The remaining constructs were mechanically tested to determine their mechanical properties. Two weeks of in vitro mechanical stimulation significantly increased collagen type I and collagen type III gene expression of the stem cell-collagen sponge constructs. Stimulated constructs showed 3 and 4 times greater collagen type I (p = 0.0001) and collagen type III gene expression (p = 0.001) than nonstimulated controls. Stimulated constructs also had 2.5 times the linear stiffness and 4 times the linear modulus of nonstimulated constructs. However, mechanical stimulation did not significantly increase decorin or fibronectin gene expression (p = 0.2) after 14 days in culture. This study shows that mechanical stimulation of cell-sponge constructs produces similar increases in the expression of 2 structural genes, as well as linear stiffness and linear modulus.
Asunto(s)
Colágeno Tipo II/metabolismo , Colágeno Tipo I/metabolismo , Expresión Génica , Mecanotransducción Celular/fisiología , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/fisiología , Animales , Células Cultivadas , Colágeno Tipo I/química , Elasticidad , Femenino , Expresión Génica/fisiología , Técnicas In Vitro , Ligamento Rotuliano/lesiones , Ligamento Rotuliano/patología , Ligamento Rotuliano/cirugía , Estimulación Física , Conejos , Estrés Mecánico , Resistencia a la TracciónRESUMEN
The objective of this study was to determine how mechanical stimulation affects the biomechanics and histology of stem cell-collagen sponge constructs used to repair central rabbit patellar tendon defects. Autogenous tissue-engineered constructs were created for both in vitro and in vivo analyses by seeding mesenchymal stem cells from 10 adult rabbits at 0.14x10(6) cells/construct in type I collagen sponges. Half of these constructs were mechanically stimulated once every 5 min for 8 h/day to a peak strain of 4% for 2 weeks. The other half remained in an incubator without mechanical stimulation for 2 weeks. Samples allocated for in vitro testing revealed that mechanically stimulated constructs had 2.5 times the linear stiffness of nonstimulated constructs. The remaining paired constructs for in vivo studies were implanted in bilateral full-thickness, full-length defects in the central third of rabbit patellar tendons. Twelve weeks after surgery, repair tissues were assigned for biomechanical (7 pairs) and histologic (3 pairs) analyses. Maximum force, linear stiffness, maximum stress, and linear modulus for the stimulated (vs. nonstimulated) repairs averaged 70% (vs. 55%), 85% (vs. 55%), 70% (vs. 50%), and 50% (vs. 40%) of corresponding values for the normal central third of the patellar tendons. The average force-elongation curve for the mechanically stimulated repairs also matched the corresponding curve for the normal patellar tendons, up to 150% of the peak in vivo force values recorded in a previous study. Construct and repair linear stiffness and linear modulus were also positively correlated (r = 0.6 and 0.7, respectively). Histologically both repairs showed excellent cellular alignment and mild staining for decorin and collagen type V, and moderate staining for fibronectin and collagen type III. This study shows that mechanical stimulation of stem cell-collagen sponge constructs can significantly improve tendon repair biomechanics up to and well beyond the functional limits of in vivo loading.
Asunto(s)
Materiales Biocompatibles , Colágeno , Trasplante de Células Madre , Células Madre/citología , Traumatismos de los Tendones/cirugía , Animales , Fenómenos Biomecánicos , Femenino , Conejos , Ingeniería de TejidosRESUMEN
The objective of the present study was to test the hypotheses that implantation of cell-seeded constructs in a rabbit Achilles tendon defect model would 1) improve repair biomechanics and matrix organization and 2) result in higher failure forces than measured in vivo forces in normal rabbit Achilles tendon (AT) during an inclined hopping activity. Autogenous tissue-engineered constructs were fabricated in culture between posts in the wells of silicone dishes at four cell-to-collagen ratios by seeding mesenchymal stem cells (MSC) from 18 adult rabbits at each of two seeding densities (0.1 x 10(6) and 1 x 10(6) cell/mL) in each of two collagen concentrations (1.3 and 2.6 mg/mL). After 5 days of contraction, constructs having the two highest ratios (0.4 and 0.8 M/mg) were damaged by excessive cell traction forces and could not be used in subsequent in vivo studies. Constructs at the lower ratios (0.04 and 0.08 M/mg) were implanted in bilateral, 2 cm long gap defects in the rabbit's lateral Achilles tendon. At 12 weeks after surgery, both repair tissues were isolated and either failed in tension (n = 13) to determine their biomechanical properties or submitted for histological analysis (n = 5). No significant differences were observed in any structural or mechanical properties or in histological appearance between the two repair conditions. However, the average maximum force and maximum stress of these repairs achieved 50 and 85% of corresponding values for the normal AT and exceeded the largest peak in vivo forces (19% of failure) previously recorded in the rabbit AT. Average stiffness and modulus were 60 and 85% of normal values, respectively. New constructs with lower cell densities and higher scaffold stiffness that do not excessively contract and tear in culture and that further improve the repair stiffness needed to withstand various levels of expected in vivo loading are currently being investigated.
Asunto(s)
Tendón Calcáneo/crecimiento & desarrollo , Colágeno/química , Trasplante de Células Madre Mesenquimatosas/métodos , Recuperación de la Función/fisiología , Traumatismos de los Tendones/fisiopatología , Ingeniería de Tejidos/métodos , Tendón Calcáneo/patología , Tendón Calcáneo/cirugía , Animales , Fenómenos Biomecánicos/métodos , Recuento de Células , Femenino , Implantes Experimentales , Modelos Anatómicos , Conejos , Traumatismos de los Tendones/etiología , Traumatismos de los Tendones/patología , Traumatismos de los Tendones/cirugía , Resultado del TratamientoRESUMEN
The objective of this study was to introduce mesenchymal stem cells (MSCs) into a gel-sponge composite and examine the effect the cells have on repair biomechanics and histology 12 weeks postsurgery. We tested two related hypotheses-adding MSCs would significantly improve repair biomechanics and cellular organization, and would result in higher failure forces than peak in vivo patellar tendon (PT) forces recorded for an inclined hopping activity. Autogenous tissue-engineered constructs were created by seeding MSCs from 15 adult rabbits at 0.1 x 10(6) cells/mL in 2.6 mg/mL of collagen gel in collagen sponges. Acellular constructs were created using the same concentration of collagen gel in matching collagen sponges. These cellular and acellular constructs were implanted in bilateral full-thickness, full-length defects in the central third of patellar tendons. At 12 weeks after surgery, repair tissues were assigned for biomechanical (n = 12 pairs) and histological (n = 3 pairs) analyses. Maximum force and maximum stress for the cellular repairs were about 60 and 50% of corresponding values for the normal central third of the PT, respectively. Likewise, linear stiffness and linear modulus for these cellular repairs averaged 75 and 30% of normal PT values, respectively. By contrast, the acellular repairs exhibited lower percentages of normal PT values for maximum force (40%), maximum stress (25%), linear stiffness (30%), and linear modulus (20%). Histologically, both repairs showed strong staining for collagen types III and V, fibronectin, and decorin. The cellular repairs also showed cellular alignment comparable to that of normal tendon. This study shows that introducing autogenous mesenchymal stem cells into a gel-collagen sponge composite significantly improves tendon repair compared to the use of a gel-sponge composite alone in the range of in vivo loading.
Asunto(s)
Colágeno/química , Trasplante de Células Madre Mesenquimatosas , Rótula/lesiones , Recuperación de la Función/fisiología , Traumatismos de los Tendones/fisiopatología , Traumatismos de los Tendones/cirugía , Animales , Fenómenos Biomecánicos , Colágeno Tipo I/química , Colágeno Tipo III/química , Femenino , Geles , Ilion/citología , Inmunohistoquímica , Implantes Experimentales , Células Madre Mesenquimatosas/citología , Conejos , Traumatismos de los Tendones/patología , Tendones/crecimiento & desarrollo , Tendones/patología , Tendones/cirugía , Factores de Tiempo , Ingeniería de Tejidos/métodos , Resultado del TratamientoRESUMEN
Autogenous tissue-engineered constructs were fabricated at four cell-to-collagen ratios (0.08, 0.04, 0.8, and 0.4 M/mg) by seeding mesenchymal stem cells (MSCs) from 16 adult rabbits at one of two seeding densities (0.1 x 10(6) and 1 x 10(6) cells/mL) in one of two collagen concentrations (1.3 and 2.6 mg/mL). The highest two ratios (0.4 and 0.8 M/mg) were damaged by excessive cell contraction and could not be used in subsequent in vivo studies. The remaining two sets of constructs were implanted into bilateral full-thickness, full-length defects created in the central third of the patellar tendon (PT). At 12 weeks after surgery, repair tissues were assigned for biomechanical (n = 13) and histological (n = 3) analyses. A second group of rabbits (n = 6) received bilateral acellular implants with the same two collagen concentrations. At 12 weeks, repair tissues were also assigned for biomechanical (n = 4) and histological (n = 2) analyses. No significant differences were observed in any structural or material properties or in histological appearance among the two cell-seeded and two acellular repair groups. Average maximum force and maximum stress of the repairs were approximately 30% of corresponding values for the central one-third of normal PT and higher than peak in vivo forces measured in rabbit PT from one of our previous publications. However, average repair stiffness and modulus were only 30 and 20% of normal PT values, respectively. Current repairs achieved higher maximum forces than in previous studies and without ectopic bone, but will need to achieve sufficient stiffness as well to be effective in the in vivo range of loading.