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.

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.

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.

Cell-monolayer-based assays for chemotherapeutic drug discovery have proven to be highly artificial compared with physiological systems. The objective of this study was to culture cancer cells in a simple 3-dimensional (3D) collagen gel model to study the antiproliferative activity of known lung cancer drugs. The validity of our 3D model was tested by measuring the activity of 10 lung cancer drugs (Paclitaxel, Alimta, Zactima, Doxorubicin, Vinorelbine, Gemcitabine, 17AAg, Cisplatin, and 2 experimental drugs from the University of Kansas [KU174 and KU363]) in 2 lung cancer cell lines (A549 and H358) and comparing the activity in a traditional 2-dimensional (2D) in vitro cellular assay. Both potency and efficacy of these drugs were calculated to evaluate the activity of the drugs. Our results demonstrate that the activity of these drugs showed significant differences when tested in 3D cultures, which varied with individual drugs and the cell line used for testing. For example, the cytotoxicity of Paclitaxel, KU174, Alimta, Zacitma, Doxorubicin, Vinorelbine, KU363, and 17AAg was significantly changed when tested in the 3D model, whereas the potency of Cisplatin and Gemcitabine in H358 cell line remained unaffected. A similar pattern, with some differences, was observed in A549 cells and is discussed in detail in this article. The observed differences in potency and efficacy of the cancer drugs in 3D models suggest that the biological implications of screening configurations should be taken into account to select superior cancer drug candidates in preclinical studies.

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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.

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 8 h=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.

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.